MIC-1 fusion proteins and uses thereof

ABSTRACT

The invention relates to MIC-1 fusion proteins. More specifically it relates to compounds comprising fusion proteins comprising a MIC-1 protein or an analog thereof at the C-terminus of the fusion protein and a functional variant of human serum albumin at the N-terminus of the fusion protein connected via a peptide linker. The compounds of the invention have MIC-1 activity. The invention also relates to pharmaceutical compositions comprising such compounds and pharmaceutically acceptable excipients, as well as the medical use of the compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/742,159 filed Jun. 17, 2015 which claims priority under 35 U.S.C. §119 to European Patent Application 14173664.5, filed Jun. 24, 2014; thecontents of which is incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to MIC-1 fusion proteins and theirpharmaceutical use.

INCORPORATION-BY-REFERENCE OF THE SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 13, 2016, isnamed 140026US01_SL_ST25.txt and is 28,566 bytes in size.

BACKGROUND

The macrophage inhibitory cytokine-1 (MIC-1), also known as GDF-15 andplacental bone morphogenetic protein (PLAB), is a distant member of theTGF-beta super family, a family of peptide hormones involved in cellgrowth and differentiation. MIC-1 circulates as a cysteine-richhomodimer with a molecular mass of 24.5 kDa. MIC-1 was initiallyreported to be up-regulated in macrophages by stimuli including IL-1b,TNF-alpha, IL-2, and TGF-b. It was also shown that MIC-1 could reducelipopolysaccharide-induced TNF-alpha production and it was based onthese data proposed that MIC-1 was an anti-inflammatory cytokine. Morerecently, a study was investigating why human patients with advancedcancer were losing body weight and they showed that the weight losscorrelated with circulating levels of MIC-1. These data indicates thatMIC-1 regulates body weight. This hypothesis was tested in micexenografted with prostate tumor cells, where data showed that elevatedMIC-1 levels were associated with loss of body weight and decreased foodintake, this effect being reversed by administration of antibodies toMIC-1. As administration of recombinant MIC-1 to mice regulatedhypothalamic neuropeptide Y and pro-opiomelanocortin it was proposedthat MIC-1 regulates food intake by a central mechanism. Furthermore,transgenic mice overexpressing MIC-1 are gaining less weight and bodyfat both on a normal low fat diet and on a high fat diet. Also,transgenic mice overexpressing MIC-1 fed both on a low and high fatdiet, respectively, had improved glucose tolerance compared with wildtype animals on a comparable diet.

Native MIC-1 has a short half-life, meaning that treatment with nativeMIC-1 requires daily administration to maintain efficacy.

WO 2001079443 concerns the use of human serum albumin or variantsthereof for fusions to peptides of pharmaceutical interest.

WO 2005099746 concerns a method of modulating appetite and/or bodyweight by administering a MIC-1 modulating agent.

SUMMARY

The invention relates to MIC-1 fusion proteins.

In one aspect, the invention provides compounds comprising fusionproteins comprising a MIC-1 protein or an analogue thereof at theC-terminus of the fusion protein and a functional variant of human serumalbumin (HSA) at the N-terminus of the fusion protein connected via apeptide linker. The peptide linker has a length of 10 to 50 amino acidsand comprises the amino acid sequence [X-Y_(m)]_(n), wherein X is Asp orGlu; Y is Ala; m is from 2 to 4, and n is at least 2.

In one aspect of the invention, Y is selected from the group of codedamino acids except for Pro and Gly. In another aspect, Y is selectedfrom the group of coded non-polar amino acids, except for Pro and Gly.

In one aspect, the invention provides a polynucleotide molecule encodinga compound comprising a fusion protein comprising a MIC-1 protein or ananalogue thereof at the C-terminus of the fusion protein and afunctional variant of HUMAN SERUM ALBUMIN at the N-terminus of thefusion protein connected via a peptide linker.

In one aspect, the invention provides a pharmaceutical compositioncomprising a compound of the invention or a pharmaceutically acceptablesalt, amide or ester thereof, and one or more pharmaceuticallyacceptable excipients.

In one aspect, the invention provides a compound of the invention foruse as a medicament.

In one aspect, the invention provides a compound of the invention foruse in the treatment of eating disorders, such as obesity, e.g. bydecreasing food intake, reducing body weight, suppressing appetite andinducing satiety.

In one aspect, the invention provides a compound of the invention foruse in the treatment of obesity.

In one aspect, the compounds of the invention are MIC-1 agonists. In oneaspect, the compounds of the invention inhibit food intake. In oneaspect, the compounds of the invention reduce body weight.

In one aspect, the compounds of the invention have longer half-life thanthe half-life of native MIC-1.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. Schematic representation of a HSA-MIC-1 dimeric fusion protein.A, B and C depicts relative positions of the human serum albumin domain,the linker region and MIC-1, respectively. —SS— indicates interchaindisulphide bridge linking together the two HSA-MIC-1 monomers to form afunctional dimeric fusion protein.

DESCRIPTION

The invention relates to compounds comprising MIC-1 fusion proteins. Inone aspect, the invention relates to MIC-1 fusion proteins.

In one aspect, the invention provides compounds comprising fusionproteins comprising MIC-1 or an analogue thereof at the C-terminus ofthe fusion protein and human serum albumin (HSA) or a functional variantthereof at the N-terminus of the fusion protein connected via a peptidelinker. The peptide linker has a length of 10 to 50 amino acids andcomprises the amino acid sequence [X-Y_(m)]_(n), wherein X is Asp orGlu; Y is Ala; m is from 2 to 4, and n is at least 2.

In one aspect of the invention, Y is selected from the group of codedamino acids except for Pro and Gly. In another aspect, Y is selectedfrom the group of coded non-polar amino acids, except for Pro and Gly.

The fusion protein strategy of the present invention combines thesoluble, stable plasma protein human serum albumin with native MIC-1 ora MIC-1 analogue. Human serum albumin has inherent properties such ashigh solubility and stability which makes it beneficial to use as fusionpartner for improving expression yield and conferring stability toMIC-1. Human serum albumin as fusion partner may also increase theplasma half-life of MIC-1 by significant size increase, which inhibitsrenal clearance and/or by binding the Fc Neonatal Receptor, which allowsrecycling from the endosome and prevention of lysomal degradationallowing the molecule to be present longer in circulation. As with othersmaller therapeutic proteins, native MIC-1 disappears rapidly from thebloodstream due to a short plasma half-life, meaning that treatment withnative MIC-1 requires daily administration to maintain efficacy. Thepresent invention provides compounds comprising MIC-1 fusion proteinswith increased plasma half-life.

In what follows, Greek letters may be represented by their symbol or thecorresponding written name, for example: α=alpha; β=beta; ϵ=epsilon;γ=gamma; ω=omega; etc. Also, the Greek letter of μ may be represented by“u”, e.g. in μl=ul, or in μM=uM.

MIC-1 Proteins and Analogues

The term “MIC-1” as used herein means macrophage inhibitory cytokine-1(MIC-1), also known as Growth Differentiation Factor 15 (GDF-15), andplacental bone morphogenetic protein (PLAB). The sequence of the fulllength wild type human MIC-1 protein is available from the UNIPROTdatabase with accession no. Q99988. The 308 amino acid precursor proteinincludes a signal peptide (amino acids 1-29), a propeptide (amino acids30-196) and a mature protein (amino acids 197-308). The 112 amino acidmature MIC-1 protein is included herein as SEQ ID NO:1. Mature MIC-1contains nine cysteine residues which give rise to the formation of 4intrachain disulphide bonds and one interchain disulphide bond to createa covalently linked 24.5 kDa homodimer. A naturally occurring mutationcorresponding to His6Asp in the mature protein (SEQ ID NO:1) has beendescribed.

Thus particular examples of wild type human MIC-1 are the mature MIC-1protein of SEQ ID NO:1, SEQ ID NO:1 having the amino acid modificationHis6Asp, as well as any of these sequences preceded by the propeptideand/or signal peptide referred to above.

The term “MIC-1 protein” as used herein refers to the human MIC-1protein of SEQ ID NO:1, or an analogue thereof. The protein having thesequence of SEQ ID NO:1 may also be designated “hMIC-1”, “native” MIC-1or “wild type” MIC-1.

The term “MIC-1 analogue”, or “analogue of MIC-1 protein” as used hereinrefers to a protein, or a compound, which is a variant of the matureMIC-1 protein (SEQ ID NO:1). In one aspect, the MIC-1 analogue is afunctional variant of the mature MIC-1 protein (SEQ ID NO:1). In oneaspect of the invention, the MIC-1 analogues display at least 85%, 90%or 95% sequence identity to native MIC-1 (SEQ ID NO:1).

In another aspect of the invention, the MIC-1 analogues comprise lessthan 17 amino acid modifications (substitutions, deletions, additions(including insertions) and any combination thereof) relative to humannative MIC-1 (SEQ ID NO:1).). As an example of a method fordetermination of the sequence identity between two analogues the twopeptides His6Asp MIC-1 and native MIC-1 are aligned. The sequenceidentity of the His6Asp MIC-1 analogue relative to native MIC-1 is givenby the number of aligned identical residues minus the number ofdifferent residues divided by the total number of residues in nativeMIC-1. Accordingly, in said example the sequence identity in percentageis (112−1)/112×100.

The term “amino acid modification” used throughout this application isused in the meaning of a modification to an amino acid as compared tonative MIC-1 (SEQ ID NO:1). This modification can be the result of adeletion of an amino acid, addition of an amino acid, substitution ofone amino acid with another or a substituent covalently attached to anamino acid of the peptide.

Substitutions.

In one aspect amino acids may be substituted by conservativesubstitution. The term “conservative substitution” as used hereindenotes that one or more amino acids are replaced by another,biologically similar residue. Examples include substitution of aminoacid residues with similar characteristics, e.g. small amino acids,acidic amino acids, polar amino acids, basic amino acids, hydrophobicamino acids and aromatic amino acids.

In one aspect amino acids may be substituted by non-conservativesubstitution. The term “non-conservative substitution” as used hereindenotes that one or more amino acids are replaced by another amino acidhaving different characteristics. Examples include substitution of abasic amino acid residue with an acidic amino acid residue, substitutionof a polar amino acid residue with an aromatic amino acid residue, etc.In one aspect, the non-conservative substitution is substitution of acoded amino acid to another coded amino acid having differentcharacteristics. In one aspect, the MIC-1 analogues may comprisesubstitutions of one or more unnatural and/or non-amino acids, e.g.,amino acid mimetics, into the sequence of MIC-1.

The asparagine residue in position 3 of human mature MIC-1 (SEQ ID NO:1)is chemically labile. In one aspect of the invention, the asparagine inthe position corresponding to position 3 of human mature MIC-1 (SEQ IDNO:1) may be substituted to Ser, Asp, Glu, Ala, Pro, Thr, Gly, or Gln.In one aspect of the invention, the asparagine in the positioncorresponding to position 3 of human mature MIC-1 (SEQ ID NO:1) has beensubstituted to Ser. In another aspect of the invention, the asparaginein the position corresponding to position 3 of human mature MIC-1 (SEQID NO:1) has been substituted to Glu.

In one aspect of the invention, the arginine in the positioncorresponding to position 2 of human mature MIC-1 (SEQ ID NO:1) has beensubstituted to alanine.

In one aspect of the invention, the arginine in the positioncorresponding to position 2 of human mature MIC-1 (SEQ ID NO:1) has beensubstituted to alanine, and the asparagine in the position correspondingto position 3 of human mature MIC-1 (SEQ ID NO:1) has been substitutedto Glu.

Deletions and Truncations. In one aspect, the MIC-1 analogues of theinvention may have one or more amino acid residues deleted from theamino acid sequence of human MIC-1, alone or in combination with one ormore insertions or substitutions.

In one aspect, the three N-terminal amino acids of human mature MIC-1(Ala1, Arg2, Asn3) may be deleted.

Insertions.

In one aspect, the MIC-1 analogues of the invention may have one or moreamino acid residues inserted into the amino acid sequence of humanMIC-1, alone or in combination with one or more deletions and/orsubstitutions.

In one aspect, the MIC-1 analogues of the invention may includeinsertions of one or more unnatural amino acids and/or non-amino acidsinto the sequence of MIC-1.

MIC-1 analogues may be described by reference to i) the number of theamino acid residue in the mature MIC-1 protein which corresponds to theamino acid residue which is changed (i.e., the corresponding position innative MIC-1), and to ii) the actual change. In other words, a MIC-1analogue is a MIC-1 protein in which a number of amino acid residueshave been changed when compared to native MIC-1 (SEQ ID NO: 1). Thesechanges may represent, independently, one or more amino acidsubstitutions, additions, and/or deletions.

As is apparent from the above examples, amino acid residues may beidentified by their full name, their one-letter code, and/or theirthree-letter code. These three ways are fully equivalent.

The term “protein”, as e.g. used in the context of MIC-1 proteins,refers to a compound which comprises a series of amino acidsinterconnected by amide (or peptide) bonds.

Amino acids are molecules containing an amine group and a carboxylicacid group, and, optionally, one or more additional groups, oftenreferred to as a side chain.

The term “amino acid” includes coded (or proteinogenic or natural) aminoacids (amongst those the 20 standard amino acids), as well as non-coded(or non-proteinogenic or non-natural) amino acids. Coded amino acids arethose which are naturally incorporated into proteins. The standard aminoacids are those encoded by the genetic code. Non-coded amino acids areeither not found in proteins, or not produced by standard cellularmachinery (e.g., they may have been subject to post-translationalmodification). In what follows, all amino acids of the MIC-1 proteinsfor which the optical isomer is not stated is to be understood to meanthe L-isomer (unless otherwise specified).

Human Serum Albumin

Human serum albumin (HSA) belongs to a family of globular proteins andis composed of 585 amino acids with an approximate molecular weight of67 kDa. Albumin comprises three homologous domains that assemble to forma heart-shaped molecule. Albumin is water-soluble and soluble inconcentrated salt solutions and is commonly found in blood plasma.Albumin is the most abundant protein of human blood plasma and its mainfunction is to regulate the osmotic pressure of blood, transporthormones or fatty acid and buffer pH. The normal range of human serumalbumin in adults is 35 to 50 g/L and human serum albumin accounts for80-90% of all plasma protein. As human serum albumin is a naturalcarrier for exogenous ligands, it has a low risk of inducing toxicityand immunogenicity and human serum albumin extracted from human bloodcan be used for clinical purposes. The plasma half-life of human serumalbumin is approximately 20 days. The long half-life of human serumalbumin is caused in part by a pH-dependent recycling mediated by theneonatal Fc receptor (FcRn). FcRn is present in cells and on the surfaceof cells, which interacts with circulating blood, such as vascularendothelial cells.

Recombinant human serum albumin fusion proteins comprising a therapeuticprotein of interest may be achieved by genetic manipulation, such thatthe DNA coding for human serum albumin, or a fragment thereof, is joinedto the DNA encoding for the therapeutic protein. A suitable expressionhost is then transformed or transfected with the fused nucleotidesequences encoded on a suitable plasmid as to express the fusionprotein. Human serum albumin as fusion partner is thought to increasethe plasma half-life of therapeutic proteins through two biologicalmechanisms. The significant size increase inhibits renal clearance andthe inherent ability of human serum albumin to bind the Fc NeonatalReceptor will allow recycling from the endosome and prevention oflysomal degradation altogether allowing the molecule to be presentlonger in circulation.

Albumin fusion proteins can be produced in expression systems on acommercial scale and with lower cost than for other methods ofgenerating therapeutic proteins with long plasma half-lives.

It is known to the person skilled in the art that functional variants ofhuman serum albumin can be designed, which have the same plasmahalf-life prolonging benefits as the wild-type (truncated and/or aminoacid substituted functional variants). For an example domain III ofhuman serum albumin has been shown to bind FcN to a high degree and itis possible to make variants comprising only this domain or combinationswith other domains, with long half-lives or half-lives that are modified(eg. Albufuse Flex Technology, Novozymes).

The sequence of the wild-type mature human serum albumin is includedherein as SEQ ID NO:2 and the sequence is annotated in the Uniprotdatabase with the accession no: P02768. The present invention provides ahuman serum albumin fusion protein comprising, or alternativelyconsisting of, a biologically active MIC-1 protein or a variant thereofand a biologically active and/or therapeutically active fragment orvariant of human serum albumin. In one aspect, the invention provides ahuman serum albumin fusion protein comprising, or alternativelyconsisting of, mature native MIC-1 and the mature native human serumalbumin. In one aspect of the invention, the primary sequence of humanserum albumin is modified. Non-limiting examples includes functionalvariants of human serum albumin comprising truncations or amino acidsubstitutions or deletions in human serum albumin, which do notinterfere with the half-life extending effect of human serum albumin.Human serum albumin contains a single thiol group from an unpairedcysteine residue at position 34 in Domain I. Cys34 in human serumalbumin provides antioxidant activity and constitutes the largestfraction of free thiol groups in the blood. Cys34-Cys34 disulfidelinkage of two human serum albumin molecules has several disadvantages,which includes side reactions with other residues during preparation,low stability or structural changes, which promotes protein aggregation.Substitutions of Cys34 with other amino acids, such as Ala or Ser hasbeen described previously (Mccurdy, T et. Al., Journal of Laboratory andClinical Medicine, Volume 143, Issue 2, 2004, 115-124). The term “HSAC34A” refers to a human serum albumin (HSA) variant wherein the cysteineresidue at position 34 of the wild type human serum albumin amino acidsequence has been replaced with alanine. Other ways of preventingdimerization and instability through unfavourable interaction of freeCys at position 34 includes truncation of the N-terminal of human serumalbumin domain I or removal of the Cys residue from the sequence.

By “functional variant” as used herein is meant a chemical variant of acertain protein which retains substantially the same function as theoriginal protein.

Fusion Proteins

“Fusion protein” as used herein is intended to mean a hybrid proteinexpressed by a nucleic acid molecule comprising nucleotide sequences ofat least two genes. “Fusion protein” as used herein is also intended tomean covalent joining of at least two proteins and/or peptides. In oneaspect, the fusion proteins of the invention comprise human serumalbumin as fusion partner fused with native MIC-1 having an activity ofpharmaceutical interest. Fusion proteins are often used for improvingrecombinant expression or stability of therapeutic proteins as well asfor improved recovery and purification of such proteins from cellcultures and the like. Fusion proteins may comprise artificialsequences, e.g. a linker sequence.

“Fusion partner” as used herein is intended to mean a protein which ispart of a fusion protein, i.e. one of the at least two proteinsencompassed by the fusion protein.

In one embodiment of the invention the fusion partner comprises humanserum albumin with an approximate molecular weight of 67 kDa (SEQ IDNO:2) or functional variants thereof, which is operatively linked to theN-terminal of MIC-1 (SEQ ID NO:1) or functional variants thereof with amolecular weight of approximately 12 kDa via an interdomain linkerregion consisting of amino acid sequences of different length, chargesand/or structural motifs.

“Fusion tag” as used herein is intended to mean a protein sequence whichis part of a fusion protein, i.e. one of the at least two proteinsencompassed by the fusion protein and comprises a sequence whichimproves expression, solubilisation or purification of the fusionprotein, e.g. a 6× Histidine tag (such as His6) or a solubilizationdomain (such as Thiol:disulfide interchange protein DsbC (DsbC), MaltoseBinding Protein (MBP), or Thioredoxin (Trx)).

In one aspect of the invention, monomers of NH2-HSA-linker-MIC-1-COOHwith a size of approximately 80 kDa, homodimerizes as the nativemolecule via interchain disulphide bridge between the two MIC-1molecules to form an active HSA-MIC-1 fusion protein with a molecularweight of approximately 160-165 kDa (depicted as schematic drawing inFIG. 1).

Peptide Linker

The term “peptide linker” as used herein is intended to mean an aminoacid sequence which is typically used to facilitate the function,folding or expression of fusion proteins.

Different exposure of the MIC-1 protein comprised in a fusion protein toits putative receptor, plasma half-life or overall fusion proteinstability may be affected by differences in the linkersequence/structure of the fusion protein, which can cause changes inbiological efficacy, plasma half-life or fusion protein stability.

The linkers from the present invention were designed with differentpredicted biophysical or structural properties comprising variations inlength (variation of the linker length), and predicted secondarystructure such as alpha-helical structure, rigid structure or flexible,random coil structures or charge. In the present invention the length ofthe linker was varied from 7 to 35 amino acids. The linker length mayinfluence the potential interaction between the human serum albumin andMIC-1 domain by changing the possibility of steric hindrance provided bythe fusion partner attached to the biological active MIC-1 domain. Thesteric hindrance may influence correct folding of the two domains of thefusion protein monomer, formation of the dimer, the interaction of theMIC-1 part with a putative receptor, or the linker itself may interactwith either human serum albumin or MIC-1 and that both composition andlength of the linker may in part influence the nature and extent of suchinteraction.

Functional Properties

Biological Activity—In Vivo Pharmacology

In one aspect the compounds of the invention are potent in vivo, whichmay be determined as is known in the art in any suitable animal model,as well as in clinical trials.

The non-obese Sprague Dawley rat is one example of a suitable animalmodel, and the changes in food intake may be determined in such rats invivo, e.g. as described in Example 2.

In one aspect the compounds of the invention inhibits in vivo foodintake in non-obese Sprague Dawley rats.

As an example, in a particular aspect of the invention, the maximumefficacy which is the greatest significant (p<0.10) reduction in 24 hourfood intake recorded over 6-7 days at a dose of 4 nmol/kg should be morethan 20%, preferably more than 30%. In another particular aspect of theinvention, the maximum efficacy which is the greatest significant(p<0.10) reduction in 24 hour food intake recorded over 6-7 days at adose of 4 nmol/kg should be at least 20%, preferably at least 30%.

As an example, in a particular aspect of the invention, the accumulatedefficacy which is the sum of significant (p<0.10) reductions in 24 hourfood intake compared with vehicle at a dose of 4 nmol/kg should be morethan 50%, more preferably more than 70%, even more preferably more than80%, or most preferably more than 100%.

As an example, in a particular aspect of the invention, the accumulatedefficacy which is the sum of significant (p<0.10) reductions in 24 hourfood intake compared with vehicle at a dose of 4 nmol/kg should be atleast 50%, more preferably at least 70%, even more preferably at least80%, or most preferably at least 100%.

Diet-Induced Obese (DIO) Sprague Dawley rats is another example of asuitable animal model, and the changes in food intake may be determinedin such rats in vivo, e.g. as described in Example 3.

In one aspect the compounds of the invention inhibits in vivo foodintake in DIO Sprague Dawley rats.

In one aspect of the invention, the maximum efficacy which is thegreatest significant (p<0.10) reduction in 24 hour food intake recordedover 6-7 days at a dose of 4 nmol/kg is at least 50%, or preferably atleast 60%.

In one aspect of the invention, the accumulated efficacy which is thesum of significant (p<0.10) reductions in 24 hour food intake comparedwith vehicle at a dose of 4 nmol/kg is at least 300%, more preferably atleast 340%, or even more preferably at least 380%.

Biophysical Properties

In one aspect, the compounds of the invention have good biophysicalproperties. These properties include but are not limited to physicalstability and/or solubility. These and other biophysical properties maybe measured using standard methods known in the art. In a particularembodiment, these properties are improved as compared to native MIC-1(SEQ ID NO:1). Increased biophysical stability of a fusion proteincompared to native MIC-1 may be at least partly be owing to stabilizingeffects of the fusion partner or the length or composition of theintervening amino acid linker inserted between the human serum albuminand MIC-1 sequence.

Production Processes

Fusion proteins such as those of the present invention may be producedby means of recombinant protein technology known to persons skilled inthe art. In general, nucleic acid sequences encoding the proteins ofinterest or functional variants thereof are modified to encode thedesired fusion protein. This modification includes the in-frame fusionof the nucleic acid sequences encoding the two or more proteins to beexpressed as a fusion protein. Such a fusion protein can be with orwithout a linker peptide as well as the fusion protein fused to a fusiontag, e.g. a Histidine tag (such as His6) or a solubilization domain(such as DsbC, MBP or Trx). This modified sequence is then inserted intoan expression vector, which is in turn transformed or transfected intothe expression host cells.

The nucleic acid construct encoding the fusion protein may suitably beof genomic, cDNA or synthetic origin. Amino acid sequence alterationsare accomplished by modification of the genetic code by well-knowntechniques.

The DNA sequence encoding the fusion protein is usually inserted into arecombinant vector which may be any vector, which may conveniently besubjected to recombinant DNA procedures, and the choice of vector willoften depend on the host cell into which it is to be introduced. Thus,the vector may be an autonomously replicating vector, i.e. a vector,which exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g. a plasmid. Alternatively,the vector may be one which, when introduced into a host cell, isintegrated into the host cell genome and replicated together with thechromosome(s) into which it has been integrated.

The vector is preferably an expression vector in which the DNA sequenceencoding the fusion protein is operably linked to additional segmentsrequired for transcription of the DNA. The term, “operably linked”indicates that the segments are arranged so that they function inconcert for their intended purposes, e.g. transcription initiates in apromoter and proceeds through the DNA sequence coding for thepolypeptide until it terminates within a terminator.

Thus, expression vectors for use in expressing the fusion protein willcomprise a promoter capable of initiating and directing thetranscription of a cloned gene or cDNA. The promoter may be any DNAsequence, which shows transcriptional activity in the host cell ofchoice and may be derived from genes encoding proteins either homologousor heterologous to the host cell.

Additionally, expression vectors for expression of the fusion proteinwill also comprise a terminator sequence, a sequence recognized by ahost cell to terminate transcription. The terminator sequence isoperably linked to the 3′ terminus of the nucleic acid sequence encodingthe polypeptide. Any terminator which is functional in the host cell ofchoice may be used in the present invention.

Expression of the fusion protein can be aimed for either intracellularexpression in the cytosol of the host cell or be directed into thesecretory pathway for extracellular expression into the growth medium.

Intracellular expression is the default pathway and requires anexpression vector with a DNA sequence comprising a promoter followed bythe DNA sequence encoding the fusion protein followed by a terminator.

To direct the fusion protein into the secretory pathway of the hostcells, a secretory signal sequence (also known as signal peptide or apre sequence) is needed as an N-terminal extension of the fusionprotein. A DNA sequence encoding the signal peptide is joined to the 5′end of the DNA sequence encoding the fusion protein in the correctreading frame. The signal peptide may be that normally associated withthe protein or may be from a gene encoding another secreted protein.

The procedures used to ligate the DNA sequences coding for the fusionprotein, the promoter, the terminator and/or secretory signal sequence,respectively, and to insert them into suitable vectors containing theinformation necessary for replication, are well known to persons skilledin the art (cf., for instance, Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor, New York, 1989).

The host cell into which the DNA sequence encoding the fusion protein isintroduced may be any cell that is capable of expressing the fusionprotein either intracellularly or extracellularly. The fusion proteinmay be produced by culturing a host cell containing a DNA sequenceencoding the fusion protein and capable of expressing the fusion proteinin a suitable nutrient medium under conditions permitting the expressionof the fusion protein. Non-limiting examples of host cells suitable forexpression of fusion proteins are: Escherichia coli, Saccharomycescerevisiae, as well as human embryonic kidney (HEK), Baby Hamster Kidney(BHK) or Chinese hamster ovary (CHO) cell lines. If posttranslationalmodifications are needed, suitable host cells include yeast, fungi,insects and higher eukaryotic cells such as mammalian cells.

Once the fusion protein has been expressed in a host organism it may berecovered and purified to the required quality by conventionaltechniques. Non-limiting examples of such conventional recovery andpurification techniques are centrifugation, solubilization, filtration,precipitation, ion-exchange chromatography, immobilized metal affinitychromatography (IMAC), Reversed phase—High Performance LiquidChromatography (RP-HPLC), gel-filtration and freeze drying.

Examples of recombinant expression and purification of fusion proteinsmay be found in e.g. Cordingley et al., 3. Virol. 1989, 63, pp5037-5045, Birch et al., Protein Expr Purif., 1995, 6, pp 609-618 and inWO2008/043847.

Examples of microbial expression and purification of fusion proteins maybe found in e.g. Chich et al, Anal. Biochem, 1995, 224, pp 245-249 andXin et al., Protein Expr. Purif. 2002, 24, pp 530-538.

Specific examples of methods of preparing a number of the compounds ofthe invention are included in the experimental part.

Mode of Administration

The term “treatment” is meant to include both the prevention andminimization of the referenced disease, disorder, or condition (i.e.,“treatment” refers to both prophylactic and therapeutic administrationof a compound of the invention or composition comprising a compound ofthe invention unless otherwise indicated or clearly contradicted bycontext.

The route of administration may be any route which effectivelytransports a compound of this invention to the desired or appropriateplace in the body, such as parenterally, for example, subcutaneously,intramuscularly or intravenously. Alternatively, a compound of thisinvention can be administered orally, pulmonary, rectally,transdermally, buccally, sublingually, or nasally.

The amount of a compound of this invention to be administered, thedetermination of how frequently to administer a compound of thisinvention, and the election of which compound or compounds of thisinvention to administer, optionally together with anotherpharmaceutically active agent, is decided in consultation with apractitioner who is familiar with the treatment of obesity and relateddisorders.

Pharmaceutical Compositions

Pharmaceutical compositions comprising a compound of the invention or apharmaceutically acceptable salt, amide, or ester thereof, and apharmaceutically acceptable excipient may be prepared as is known in theart.

The term “excipient” broadly refers to any component other than theactive therapeutic ingredient(s). The excipient may be an inertsubstance, an inactive substance, and/or a not medicinally activesubstance.

The excipient may serve various purposes, e.g. as a carrier, vehicle,diluent, tablet aid, and/or to improve administration, and/or absorptionof the active substance.

The formulation of pharmaceutically active ingredients with variousexcipients is known in the art, see e.g. Remington: The Science andPractice of Pharmacy (e.g. 19^(th) edition (1995), and any latereditions).

The term “physical stability” refers to the tendency of the polypeptideto form biologically inactive and/or insoluble aggregates as a result ofexposure to thermo-mechanical stress, and/or interaction withdestabilising interfaces and surfaces (such as hydrophobic surfaces).The physical stability of an aqueous polypeptide formulation may beevaluated by means of visual inspection, and/or by turbiditymeasurements after exposure to mechanical/physical stress (e.g.agitation) at different temperatures for various time periods.Alternatively, the physical stability may be evaluated using aspectroscopic agent or probe of the conformational status of thepolypeptide such as e.g. Thioflavin T or “hydrophobic patch” probes.

The term “chemical stability” refers to chemical (in particularcovalent) changes in the polypeptide structure leading to formation ofchemical degradation products potentially having a reduced biologicalpotency, and/or increased immunogenic effect as compared to the intactpolypeptide. The chemical stability can be evaluated by measuring theamount of chemical degradation products at various time-points afterexposure to different environmental conditions, e.g. by SEC-HPLC, and/orRP-HPLC.

Combination Treatment

The treatment with a compound according to the present invention mayalso be combined with one or more pharmacologically active substances,e.g., selected from antiobesity agents, appetite regulating agents, andagents for the treatment and/or prevention of complications anddisorders resulting from or associated with obesity.

Pharmaceutical Indications

In one aspect, the present invention relates to a compound of theinvention, for use as a medicament.

In particular embodiments, the compound of the invention may be used forthe following medical treatments:

(i) Prevention and/or treatment of eating disorders, such as obesity,e.g. by decreasing food intake, reducing body weight, suppressingappetite and inducing satiety.

(ii) Prevention and/or treatment of hyperglycemia and/or impairedglucose tolerance.

In some embodiments the invention relates to a method for weightmanagement. In some embodiments the invention relates to a method forreduction of appetite. In some embodiments the invention relates to amethod for reduction of food intake.

Generally, all subjects suffering from obesity are also considered to besuffering from overweight. In some embodiments the invention relates toa method for treatment or prevention of obesity. In some embodiments theinvention relates to use of the MIC-1 fusion proteins of the inventionfor treatment or prevention of obesity. In some embodiments the subjectsuffering from obesity is human, such as an adult human or a paediatrichuman (including infants, children, and adolescents). Body mass index(BMI) is a measure of body fat based on height and weight. The formulafor calculation is BMI=weight in kilograms/height in meters2. A humansubject suffering from obesity may have a BMI of ≥30; this subject mayalso be referred to as obese. In some embodiments the human subjectsuffering from obesity may have a BMI of ≥35 or a BMI in the range of≥30 to <40. In some embodiments the obesity is severe obesity or morbidobesity, wherein the human subject may have a BMI of ≥40.

In some embodiments the invention relates to a method for treatment orprevention of overweight, optionally in the presence of at least oneweight-related comorbidity. In some embodiments the invention relates touse of the MIC-1 fusion proteins of the invention for treatment orprevention of overweight, optionally in the presence of at least oneweight-related comorbidity.

In some embodiments the subject suffering from overweight is human, suchas an adult human or a paediatric human (including infants, children,and adolescents). In some embodiments a human subject suffering fromoverweight may have a BMI of ≥25, such as a BMI of ≥27. In someembodiments a human subject suffering from overweight has a BMI in therange of 25 to <30 or in the range of 27 to <30. In some embodiments theweight-related comorbidity is selected from the group consisting ofhypertension, diabetes (such as type 2 diabetes), dyslipidaemia, highcholesterol, and obstructive sleep apnoea.

In some embodiments the invention relates to a method for reduction ofbody weight. In some embodiments the invention relates to use of theMIC-1 fusion proteins of the invention for reduction of body weight. Ahuman to be subjected to reduction of body weight according to thepresent invention may have a BMI of ≥25, such as a BMI of 27 or a BMI of≥30. In some embodiments the human to be subjected to reduction of bodyweight according to the present invention may have a BMI of ≥35 or a BMIof ≥40. The term “reduction of body weight” may include treatment orprevention of obesity and/or overweight.

PARTICULAR EMBODIMENTS

The invention is further described by the following non-limitingembodiments of the invention:

1. A compound comprising a fusion protein of formula (I):A-B-C  (I),whereinA is human serum albumin or a functional variant thereof;B is a peptide linker comprising the amino acid sequence [X-Y_(m)]_(n),wherein X is Asp or Glu; Y is Ala; m is from 2 to 4; and n is at least2; andC is a MIC-1 protein or an analogue thereof, andwherein the C-terminus of human serum albumin or a functional variantthereof is fused to the N-terminus of the peptide linker, and theC-terminus of the peptide linker is fused to the N-terminus of the MIC-1protein or analogue thereof.2. A compound consisting of a fusion protein of formula (I):A-B-C  (I),whereinA is human serum albumin or a functional variant thereof;B is a peptide linker comprising the amino acid sequence [X-Y_(m)]_(n),wherein X is Asp or Glu; Y is Ala; m is from 2 to 4; and n is at least2; andC is a MIC-1 protein or an analogue thereof, andwherein the C-terminus of human serum albumin or a functional variantthereof is fused to the N-terminus of the peptide linker, and theC-terminus of the peptide linker is fused to the N-terminus of the MIC-1protein or analogue thereof.3. A compound comprising a fusion protein of formula (I):A-B-C  (I),whereinA is human serum albumin or a functional variant thereof;B is a peptide linker, wherein the peptide linker is 10 to 50 aminoacids in length and comprises the amino acid sequence [X-Y_(m)]_(n),wherein X is Asp or Glu; Y is Ala; m is from 2 to 4; and n is at least2; andC is a MIC-1 protein or an analogue thereof, andwherein the C-terminus of human serum albumin or a functional variantthereof is fused to the N-terminus of the peptide linker, and theC-terminus of the peptide linker is fused to the N-terminus of the MIC-1protein or analogue thereof.4. A compound consisting of a fusion protein of formula (I):A-B-C  (I),whereinA is human serum albumin or a functional variant thereof;B is a peptide linker, wherein the peptide linker is 10 to 50 aminoacids in length and comprises the amino acid sequence [X-Y_(m)]_(n),wherein X is Asp or Glu; Y is Ala; m is from 2 to 4; and n is at least2; andC is a MIC-1 protein or an analogue thereof, andwherein the C-terminus of human serum albumin or a functional variantthereof is fused to the N-terminus of the peptide linker, and theC-terminus of the peptide linker is fused to the N-terminus of the MIC-1protein or analogue thereof.5. A compound according to any one of the preceding embodiments, whereinthe compound is a homodimer of two fusion proteins of formula (I):A-B-C  (I)formed by an interchain disulphide bridge between the two MIC-1 proteinsor analogues thereof.6. A compound according to any one of the preceding embodiments, whereinthe peptide linker is 10 to 35 amino acids in length.7. A compound according to any one of the preceding embodiments, whereinthe peptide linker is 15 to 25 amino acids in length.8. A compound according to any one of the preceding embodiments, whereinthe peptide linker is 20 to 25 amino acids in length.8a. A compound according to any one of the preceding embodiments,wherein the peptide linker is 20 to 30 amino acids in length.9. A compound according to any one of the preceding embodiments, whereinthe peptide linker consists of a maximum of 35 amino acids.10. A compound according to any one of the preceding embodiments,wherein the peptide linker consists of a maximum of 30 amino acids.11. A compound according to any one of the preceding embodiments,wherein the peptide linker consists of a maximum of 25 amino acids.12. A compound according to any one of the preceding embodiments,wherein the peptide linker comprises the amino acid sequence[X-Y_(m)]_(n)X, wherein X is Asp or Glu; Y is Ala; m is from 2 to 4; andn is at least 2.13. A compound according to any one of the preceding embodiments,wherein the peptide linker has the amino acid sequence [X-Y_(m)]_(n)X,wherein X is Asp or Glu; Y is Ala; m is from 2 to 4; and n is at least2.14. A compound according to any one of the preceding embodiments,wherein the peptide linker has the amino acid sequence [X-Y_(m)]_(n)X,wherein X is Asp or Glu; Y is Ala; m is from 2 to 4; and n is at least5.15. A compound according to any one of the preceding embodiments,wherein the peptide linker has the amino acid sequence [X-Y_(m)]_(n)X,wherein X is Asp or Glu; Y is Ala; m is from 2 to 3; and n is at least5.16. A compound according to any one of the preceding embodiments,wherein the peptide linker comprises the amino acid sequenceGGSS[X-Y_(m)]_(n)X, wherein X is Asp or Glu; Y is Ala; m is from 2 to 4;and n is at least 2 (wherein GGSS is SEQ ID NO: 40).17. A compound according to any one of the preceding embodiments,wherein the peptide linker has the amino acid sequenceGGSS[X-Y_(m)]_(n)X, wherein X is Asp or Glu; Y is Ala; m is from 2 to 4;and n is at least 2 (wherein GGSS is SEQ ID NO: 40).18. A compound according to any one of the preceding embodiments,wherein the peptide linker has the amino acid sequenceGGSS[X-Y_(m)]_(n)X, wherein X is Asp or Glu; Y is Ala; m is from 2 to 4;and n is at least 5 (wherein GGSS is SEQ ID NO: 40).19. A compound according to any one of the preceding embodiments,wherein the peptide linker has the amino acid sequenceGGSS[X-Y_(m)]_(n)X, wherein X is Asp or Glu; Y is Ala; m is from 2 to 3;and n is at least 5 (wherein GGSS is SEQ ID NO: 40).20. A compound according to any one of the preceding embodiments,wherein the peptide linker has the amino acid sequenceGGSS[X-Y_(m)]_(n)X, wherein X is Asp or Glu; Y is Ala; m is 2; and n is5 or 6 (wherein GGSS is SEQ ID NO: 40).21. A compound according to any one of the preceding embodiments,wherein the peptide linker has the amino acid sequenceGGSS[X-Y_(m)]_(n)X, wherein X is Asp or Glu; Y is Ala; m is 2; and n is6 (wherein GGSS is SEQ ID NO: 40).22. A compound according to any one of the preceding embodiments,wherein X is Asp.23. A compound according to any one of the preceding embodiments,wherein X is Glu.24. A compound according to any one of the preceding embodiments,wherein m is 2 and n is 2, 4 or 6.25. A compound according to any one of the preceding embodiments,wherein n is 2, 4 or 6.26. A compound according to any one of the preceding embodiments,wherein n is 6.27. A compound according to any one of the preceding embodiments,wherein m is 2.27a. A compound according to any one of the preceding embodiments,wherein m is 3.28. A compound according to any one of the preceding embodiments,wherein the peptide linker comprises (Glu-Ala-Ala)₆ (SEQ ID NO: 39).29. A compound according to any one of the preceding embodiments,wherein the peptide linker is (Glu-Ala-Ala)₆ (SEQ ID NO: 39).30. A compound according to any one of the preceding embodiments,wherein the peptide linker comprises (Glu-Ala-Ala)₆-Glu (SEQ ID NO: 11).31. A compound according to any one of the preceding embodiments,wherein the peptide linker is (Glu-Ala-Ala)₆-Glu (SEQ ID NO: 11).32. A compound according to any one of the preceding embodiments,wherein the peptide linker comprises Gly-Gly-Ser-Ser-(Glu-Ala-Ala)₆-Glu(SEQ ID NO: 9).33. A compound according to any one of the preceding embodiments,wherein the peptide linker is Gly-Gly-Ser-Ser-(Glu-Ala-Ala)₆-Glu (SEQ IDNO: 9).34. A compound according to any one of the preceding embodiments,wherein the peptide linker comprises (Glu-Ala-Ala)₁₀-Glu (SEQ ID NO:12).35. A compound according to any one of the preceding embodiments,wherein the peptide linker is (Glu-Ala-Ala)₁₀-Glu (SEQ ID NO: 12).36. A compound according to any one of the preceding embodiments,wherein the peptide linker comprises Gly-Gly-Ser-Ser-(Glu-Ala-Ala)₁₀-Glu(SEQ ID NO: 13).37. A compound according to any one of the preceding embodiments,wherein the peptide linker is Gly-Gly-Ser-Ser-(Glu-Ala-Ala)₁₀-Glu (SEQID NO: 13).38. A compound according to any one of the preceding embodiments,wherein the peptide linker comprises (Glu-Ala-Ala-Ala)₅-Glu (SEQ ID NO:33).39. A compound according to any one of the preceding embodiments,wherein the peptide linker is (Glu-Ala-Ala-Ala)₅-Glu (SEQ ID NO: 33).40. A compound according to any one of the preceding embodiments,wherein the peptide linker comprisesGly-Gly-Ser-Ser-(Glu-Ala-Ala-Ala)₅-Glu (SEQ ID NO: 35).41. A compound according to any one of the preceding embodiments,wherein the peptide linker is Gly-Gly-Ser-Ser-(Glu-Ala-Ala-Ala)₅-Glu(SEQ ID NO: 35).42. A compound according to any one of the preceding embodiments,wherein the peptide linker comprises (Glu-Ala-Ala-Ala)₆-Glu (SEQ ID NO:36).43. A compound according to any one of the preceding embodiments,wherein the peptide linker is (Glu-Ala-Ala-Ala)₆-Glu (SEQ ID NO: 36).44. A compound according to any one of the preceding embodiments,wherein the peptide linker comprisesGly-Gly-Ser-Ser-(Glu-Ala-Ala-Ala)₆-Glu (SEQ ID NO: 38).45. A compound according to any one of the preceding embodiments,wherein the peptide linker is Gly-Gly-Ser-Ser-(Glu-Ala-Ala-Ala)₆-Glu(SEQ ID NO: 38).46. A compound according to any one of the preceding embodiments,wherein the peptide linker comprises (Asp-Ala-Ala)₆-Asp (SEQ ID NO: 10).47. A compound according to any one of the preceding embodiments,wherein the peptide linker is (Asp-Ala-Ala)₆-Asp (SEQ ID NO: 10).48. A compound according to any one of the preceding embodiments,wherein the peptide linker comprises (Asp-Ala-Ala-Ala)₅-Asp (SEQ ID NO:34).49. A compound according to any one of the preceding embodiments,wherein the peptide linker is (Asp-Ala-Ala-Ala)₅-Asp (SEQ ID NO: 34).50. A compound according to any one of the preceding embodiments,wherein the peptide linker comprises (Asp-Ala-Ala-Ala)₆-Asp (SEQ ID NO:37).51. A compound according to any one of the preceding embodiments,wherein C is an analogue of MIC-1 displaying at least 85% sequenceidentity to native MIC-1 (SEQ ID NO:1).52. A compound according to any one of the preceding embodiments,wherein C is an analogue of MIC-1 displaying at least 90% sequenceidentity to native MIC-1 (SEQ ID NO:1).53. A compound according to any one of the preceding embodiments,wherein C is an analogue of MIC-1 displaying at least 95% sequenceidentity to native MIC-1 (SEQ ID NO:1).54. A compound according to any one of the preceding embodiments,wherein C is an analogue of MIC-1 having a maximum of 17 amino acidmodifications compared to native MIC-1 (SEQ ID NO:1).55. A compound according to any one of the preceding embodiments,wherein C is an analogue of MIC-1 having a maximum of 11 amino acidmodifications compared to native MIC-1 (SEQ ID NO:1).56. A compound according to any one of the preceding embodiments,wherein C is an analogue of MIC-1 having a maximum of 5 amino acidmodifications compared to native MIC-1 (SEQ ID NO:1).57. A compound according to any one of the preceding embodiments,wherein C is mature human MIC-1 (SEQ ID NO:1).58. A compound according to any one of the preceding embodiments,wherein C is N3S hMIC-1 of SEQ ID NO:14.59. A compound according to any one of the preceding embodiments,wherein C is R2A, N3E hMIC-1 of SEQ ID NO:15.60. A compound according to any one of the preceding embodiments,wherein C is N3E hMIC-1 of SEQ ID NO:16.61. A compound according to any one of the preceding embodiments,wherein C is N3A hMIC-1 of SEQ ID NO:17.62. A compound according to any one of the preceding embodiments,wherein C is N3P hMIC-1 of SEQ ID NO:18.63. A compound according to any one of the preceding embodiments,wherein C is N3T hMIC-1 of SEQ ID NO:19.64. A compound according to any one of the preceding embodiments,wherein C is N3G hMIC-1 of SEQ ID NO:20.65. A compound according to any one of the preceding embodiments,wherein C is N3Q hMIC-1 of SEQ ID NO:21.66. A compound according to any one of the preceding embodiments,wherein C is N3D hMIC-1 of SEQ ID NO:22.67. A compound according to any one of the preceding embodiments,wherein C is SEQ ID NO:14.68. A compound according to any one of the preceding embodiments,wherein C is SEQ ID NO:15.69. A compound according to any one of the preceding embodiments,wherein C is SEQ ID NO:16.70. A compound according to any one of the preceding embodiments,wherein C is SEQ ID NO:17.71. A compound according to any one of the preceding embodiments,wherein C is SEQ ID NO:18.72. A compound according to any one of the preceding embodiments,wherein C is SEQ ID NO:19.73. A compound according to any one of the preceding embodiments,wherein C is SEQ ID NO:20.74. A compound according to any one of the preceding embodiments,wherein C is SEQ ID NO:21.75. A compound according to any one of the preceding embodiments,wherein C is SEQ ID NO:22.76. A compound according to any one of the preceding embodiments,wherein A is wild type human serum albumin of SEQ ID NO:2.77. A compound according to any one of the preceding embodiments,wherein A is an analogue of human serum albumin displaying at least 85%sequence identity to wild type human serum albumin of SEQ ID NO:2.78. A compound according to any one of the preceding embodiments,wherein A is an analogue of human serum albumin displaying at least 90%sequence identity to wild type human serum albumin of SEQ ID NO:2.79. A compound according to any one of the preceding embodiments,wherein A is an analogue of human serum albumin displaying at least 95%sequence identity to wild type human serum albumin of SEQ ID NO:2.80. A compound according to any one of the preceding embodiments,wherein A is C34A human serum albumin of SEQ ID NO:23.81. A compound according to any one of the preceding embodiments,wherein A is SEQ ID NO:23.82. A compound according to any one of the preceding embodiments,further comprising a fusion partner.83. A compound according to any one of the preceding embodiments,further comprising an N-terminal fusion partner.84. A compound according to any one of the preceding embodiments,wherein said compound is a MIC-1 agonist.85. A compound according to any one of the preceding embodiments,wherein said compound is capable of decreasing food intake.86. A compound according to any one of the preceding embodiments,wherein said compound has the effect in vivo of decreasing food intakedetermined in a single-dose study in non-obese Sprague Dawley rats.87. A compound according to embodiment 1, consisting of a fusion proteinof formula (I):A-B-C  (I),selected from the following:A is human serum albumin protein of SEQ ID NO:2, B is the peptide linkerof SEQ ID NO:10, and C is native human MIC-1 of SEQ ID NO:1;A is human serum albumin protein of SEQ ID NO:2, B is the peptide linkerof SEQ ID NO:11, and C is native human MIC-1 of SEQ ID NO:1;A is human serum albumin protein of SEQ ID NO:2, B is the peptide linkerof SEQ ID NO:33, and C is native human MIC-1 of SEQ ID NO:1;A is human serum albumin protein of SEQ ID NO:2, B is the peptide linkerof SEQ ID NO:34, and C is native human MIC-1 of SEQ ID NO:1;A is human serum albumin protein of SEQ ID NO:2, B is the peptide linkerof SEQ ID NO:9, and C is native human MIC-1 of SEQ ID NO:1;A is human serum albumin protein of SEQ ID NO:2, B is the peptide linkerof SEQ ID NO:35, and C is native human MIC-1 of SEQ ID NO:1;A is human serum albumin protein of SEQ ID NO:2, B is the peptide linkerof SEQ ID NO:36, and C is native human MIC-1 of SEQ ID NO:1;A is human serum albumin protein of SEQ ID NO:2, B is the peptide linkerof SEQ ID NO:37, and C is native human MIC-1 of SEQ ID NO:1;A is human serum albumin protein of SEQ ID NO:2, B is the peptide linkerof SEQ ID NO:38, and C is native human MIC-1 of SEQ ID NO:1;A is human serum albumin protein of SEQ ID NO:2, B is the peptide linkerof SEQ ID NO:12, and C is native human MIC-1 of SEQ ID NO:1;A is human serum albumin protein of SEQ ID NO:2, B is the peptide linkerof SEQ ID NO:13, and C is native human MIC-1 of SEQ ID NO:1;A is human serum albumin protein of SEQ ID NO:23, B is the peptidelinker of SEQ ID NO:9, and C is native human MIC-1 of SEQ ID NO:1;A is human serum albumin protein of SEQ ID NO:23, B is the peptidelinker of SEQ ID NO:9, and C is the MIC-1 variant of SEQ ID NO:14;A is human serum albumin protein of SEQ ID NO:23, B is the peptidelinker of SEQ ID NO:9, and C is the MIC-1 variant of SEQ ID NO:15;A is human serum albumin protein of SEQ ID NO:23, B is the peptidelinker of SEQ ID NO:9, and C is the MIC-1 variant of SEQ ID NO:16;A is human serum albumin protein of SEQ ID NO:23, B is the peptidelinker of SEQ ID NO:9, and C is the MIC-1 variant of SEQ ID NO:17;A is human serum albumin protein of SEQ ID NO:23, B is the peptidelinker of SEQ ID NO:9, and C is the MIC-1 variant of SEQ ID NO:18;A is human serum albumin protein of SEQ ID NO:23, B is the peptidelinker of SEQ ID NO:9, and C is the MIC-1 variant of SEQ ID NO:19;A is human serum albumin protein of SEQ ID NO:23, B is the peptidelinker of SEQ ID NO:9, and C is the MIC-1 variant of SEQ ID NO:20;A is human serum albumin protein of SEQ ID NO:23, B is the peptidelinker of SEQ ID NO:9, and C is the MIC-1 variant of SEQ ID NO:21; andA is human serum albumin protein of SEQ ID NO:23, B is the peptidelinker of SEQ ID NO:9, and C is the MIC-1 variant of SEQ ID NO:22.88. A compound according to embodiment 1, consisting of a fusion proteinof formula (I):A-B-C  (I),selected from the following:A is human serum albumin protein of SEQ ID NO:2, B is the peptide linkerof SEQ ID NO:9, and C is native human MIC-1 of SEQ ID NO:1;A is human serum albumin protein of SEQ ID NO:23, B is the peptidelinker of SEQ ID NO:9, and C is native human MIC-1 of SEQ ID NO:1;A is human serum albumin protein of SEQ ID NO:23, B is the peptidelinker of SEQ ID NO:9, and C is the MIC-1 variant of SEQ ID NO:14; andA is human serum albumin protein of SEQ ID NO:23, B is the peptidelinker of SEQ ID NO:9, and C is the MIC-1 variant of SEQ ID NO:15.89. A compound according to embodiment 1, wherein A is human serumalbumin protein of SEQ ID NO:2, B is the peptide linker of SEQ ID NO:10,and C is native human MIC-1 of SEQ ID NO:1.90. A compound according to embodiment 1, wherein A is human serumalbumin protein of SEQ ID NO:2, B is the peptide linker of SEQ ID NO:11,and C is native human MIC-1 of SEQ ID NO:1.91. A compound according to embodiment 1, wherein A is human serumalbumin protein of SEQ ID NO:2, B is the peptide linker of SEQ ID NO:33,and C is native human MIC-1 of SEQ ID NO:1.92. A compound according to embodiment 1, wherein A is human serumalbumin protein of SEQ ID NO:2, B is the peptide linker of SEQ ID NO:34,and C is native human MIC-1 of SEQ ID NO:1.93. A compound according to embodiment 1, wherein A is human serumalbumin protein of SEQ ID NO:2, B is the peptide linker of SEQ ID NO:9,and C is native human MIC-1 of SEQ ID NO:1.94. A compound according to embodiment 1, wherein A is human serumalbumin protein of SEQ ID NO:2, B is the peptide linker of SEQ ID NO:35,and C is native human MIC-1 of SEQ ID NO:1.95. A compound according to embodiment 1, wherein A is human serumalbumin protein of SEQ ID NO:2, B is the peptide linker of SEQ ID NO:36,and C is native human MIC-1 of SEQ ID NO:1.96. A compound according to embodiment 1, wherein A is human serumalbumin protein of SEQ ID NO:2, B is the peptide linker of SEQ ID NO:37,and C is native human MIC-1 of SEQ ID NO:1.97. A compound according to embodiment 1, wherein A is human serumalbumin protein of SEQ ID NO:2, B is the peptide linker of SEQ ID NO:38,and C is native human MIC-1 of SEQ ID NO:1.98. A compound according to embodiment 1, wherein A is human serumalbumin protein of SEQ ID NO:2, B is the peptide linker of SEQ ID NO:12,and C is native human MIC-1 of SEQ ID NO:1.99. A compound according to embodiment 1, wherein A is human serumalbumin protein of SEQ ID NO:2, B is the peptide linker of SEQ ID NO:13,and C is native human MIC-1 of SEQ ID NO:1.100. A compound according to embodiment 1, wherein A is human serumalbumin protein of SEQ ID NO:23, B is the peptide linker of SEQ ID NO:9,and C is native human MIC-1 of SEQ ID NO:1.101. A compound according to embodiment 1, wherein A is human serumalbumin protein of SEQ ID NO:23, B is the peptide linker of SEQ ID NO:9,and C is the MIC-1 variant of SEQ ID NO:14.102. A compound according to embodiment 1, wherein A is human serumalbumin protein of SEQ ID NO:23, B is the peptide linker of SEQ ID NO:9,and C is the MIC-1 variant of SEQ ID NO:15.103. A compound according to embodiment 1, wherein A is human serumalbumin protein of SEQ ID NO:23, B is the peptide linker of SEQ ID NO:9,and C is the MIC-1 variant of SEQ ID NO:16.104. A compound according to embodiment 1, wherein A is human serumalbumin protein of SEQ ID NO:23, B is the peptide linker of SEQ ID NO:9,and C is the MIC-1 variant of SEQ ID NO:17.105. A compound according to embodiment 1, wherein A is human serumalbumin protein of SEQ ID NO:23, B is the peptide linker of SEQ ID NO:9,and C is the MIC-1 variant of SEQ ID NO:18.106. A compound according to embodiment 1, wherein A is human serumalbumin protein of SEQ ID NO:23, B is the peptide linker of SEQ ID NO:9,and C is the MIC-1 variant of SEQ ID NO:19.107. A compound according to embodiment 1, wherein A is human serumalbumin protein of SEQ ID NO:23, B is the peptide linker of SEQ ID NO:9,and C is the MIC-1 variant of SEQ ID NO:20.108. A compound according to embodiment 1, wherein A is human serumalbumin protein of SEQ ID NO:23, B is the peptide linker of SEQ ID NO:9,and C is the MIC-1 variant of SEQ ID NO:21.109. A compound according to embodiment 1, wherein A is human serumalbumin protein of SEQ ID NO:23, B is the peptide linker of SEQ ID NO:9,and C is the MIC-1 variant of SEQ ID NO:22.110. A compound according to embodiment 1, consisting of a fusionprotein of formula (I):A-B-C  (I),wherein A is human serum albumin protein of SEQ ID NO:2, B is thepeptide linker of SEQ ID NO:10, and C is native human MIC-1 of SEQ IDNO:1.111. A compound according to embodiment 1, consisting of a fusionprotein of formula (I):A-B-C  (I),wherein A is human serum albumin protein of SEQ ID NO:2, B is thepeptide linker of SEQ ID NO:11, and C is native human MIC-1 of SEQ IDNO:1.112. A compound according to embodiment 1, consisting of a fusionprotein of formula (I):A-B-C  (I),wherein A is human serum albumin protein of SEQ ID NO:2, B is thepeptide linker of SEQ ID NO:33, and C is native human MIC-1 of SEQ IDNO:1.113. A compound according to embodiment 1, consisting of a fusionprotein of formula (I):A-B-C  (I),wherein A is human serum albumin protein of SEQ ID NO:2, B is thepeptide linker of SEQ ID NO:34, and C is native human MIC-1 of SEQ IDNO:1.114. A compound according to embodiment 1, consisting of a fusionprotein of formula (I):A-B-C  (I),wherein A is human serum albumin protein of SEQ ID NO:2, B is thepeptide linker of SEQ ID NO:9, and C is native human MIC-1 of SEQ IDNO:1.115. A compound according to embodiment 1, consisting of a fusionprotein of formula (I):A-B-C  (I),wherein A is human serum albumin protein of SEQ ID NO:2, B is thepeptide linker of SEQ ID NO:35, and C is native human MIC-1 of SEQ IDNO:1.116. A compound according to embodiment 1, consisting of a fusionprotein of formula (I):A-B-C  (I),wherein A is human serum albumin protein of SEQ ID NO:2, B is thepeptide linker of SEQ ID NO:36, and C is native human MIC-1 of SEQ IDNO:1.117. A compound according to embodiment 1, consisting of a fusionprotein of formula (I):A-B-C  (I),wherein A is human serum albumin protein of SEQ ID NO:2, B is thepeptide linker of SEQ ID NO:37, and C is native human MIC-1 of SEQ IDNO:1.118. A compound according to embodiment 1, consisting of a fusionprotein of formula (I):A-B-C  (I),wherein A is human serum albumin protein of SEQ ID NO:2, B is thepeptide linker of SEQ ID NO:38, and C is native human MIC-1 of SEQ IDNO:1.119. A compound according to embodiment 1, consisting of a fusionprotein of formula (I):A-B-C  (I),wherein A is human serum albumin protein of SEQ ID NO:2, B is thepeptide linker of SEQ ID NO:12, and C is native human MIC-1 of SEQ IDNO:1.120. A compound according to embodiment 1, consisting of a fusionprotein of formula (I):A-B-C  (I),wherein A is human serum albumin protein of SEQ ID NO:2, B is thepeptide linker of SEQ ID NO:13, and C is native human MIC-1 of SEQ IDNO:1.121. A compound according to embodiment 1, consisting of a fusionprotein of formula (I):A-B-C  (I),wherein A is human serum albumin protein of SEQ ID NO:23, B is thepeptide linker of SEQ ID NO:9, and C is native human MIC-1 of SEQ IDNO:1.122. A compound according to embodiment 1, consisting of a fusionprotein of formula (I):A-B-C  (I),wherein A is human serum albumin protein of SEQ ID NO:23, B is thepeptide linker of SEQ ID NO:9, and C is the MIC-1 variant of SEQ IDNO:14.123. A compound according to embodiment 1, consisting of a fusionprotein of formula (I):A-B-C  (I),wherein A is human serum albumin protein of SEQ ID NO:23, B is thepeptide linker of SEQ ID NO:9, and C is the MIC-1 variant of SEQ IDNO:15.124. A compound according to embodiment 1, consisting of a fusionprotein of formula (I):A-B-C  (I),wherein A is human serum albumin protein of SEQ ID NO:23, B is thepeptide linker of SEQ ID NO:9, and C is the MIC-1 variant of SEQ IDNO:16.125. A compound according to embodiment 1, consisting of a fusionprotein of formula (I):A-B-C  (I),wherein A is human serum albumin protein of SEQ ID NO:23, B is thepeptide linker of SEQ ID NO:9, and C is the MIC-1 variant of SEQ IDNO:17.126. A compound according to embodiment 1, consisting of a fusionprotein of formula (I):A-B-C  (I),wherein A is human serum albumin protein of SEQ ID NO:23, B is thepeptide linker of SEQ ID NO:9, and C is the MIC-1 variant of SEQ IDNO:18.127. A compound according to embodiment 1, consisting of a fusionprotein of formula (I):A-B-C  (I),wherein A is human serum albumin protein of SEQ ID NO:23, B is thepeptide linker of SEQ ID NO:9, and C is the MIC-1 variant of SEQ IDNO:19.128. A compound according to embodiment 1, consisting of a fusionprotein of formula (I):A-B-C  (I),wherein A is human serum albumin protein of SEQ ID NO:23, B is thepeptide linker of SEQ ID NO:9, and C is the MIC-1 variant of SEQ IDNO:20.129. A compound according to embodiment 1, consisting of a fusionprotein of formula (I):A-B-C  (I),wherein A is human serum albumin protein of SEQ ID NO:23, B is thepeptide linker of SEQ ID NO:9, and C is the MIC-1 variant of SEQ IDNO:21.130. A compound according to embodiment 1, consisting of a fusionprotein of formula (I):A-B-C  (I),wherein A is human serum albumin protein of SEQ ID NO:23, B is thepeptide linker of SEQ ID NO:9, and C is the MIC-1 variant of SEQ IDNO:22.131. A compound according to embodiment 1, selected from the following:compound 23, compound 24, and compound 26.132. A compound according to embodiment 1, wherein the compound iscompound 23.133. A compound according to embodiment 1, wherein the compound iscompound 24.134. A compound according to embodiment 1, wherein the compound iscompound 25.135. A compound according to embodiment 1, wherein the compound iscompound 26.136. A compound according to embodiment 1, consisting of a His-taggedfusion protein of formula (I), wherein the His-tag is the His-tag of SEQID NO:3, A is human serum albumin protein of SEQ ID NO:2, B is thepeptide linker of SEQ ID NO:9, and C is native human MIC-1 of SEQ IDNO:1.137. A compound according to embodiment 1, consisting of a His-taggedfusion protein of formula (I), wherein the His-tag is the His-tag of SEQID NO:3, A is human serum albumin protein of SEQ ID NO:2, B is thepeptide linker of SEQ ID NO:10, and C is native human MIC-1 of SEQ IDNO:1.138. A compound according to embodiment 1, consisting of a His-taggedfusion protein of formula (I), wherein the His-tag is the His-tag of SEQID NO:3, wherein A is human serum albumin protein of SEQ ID NO:2, B isthe peptide linker of SEQ ID NO:11, and C is native human MIC-1 of SEQID NO:1.139. A compound according to embodiment 1, consisting of a His-taggedfusion protein of formula (I), wherein the His-tag is the His-tag of SEQID NO:3, wherein A is human serum albumin protein of SEQ ID NO:2, B isthe peptide linker of SEQ ID NO:12, and C is native human MIC-1 of SEQID NO:1.140. A compound according to embodiment 1, consisting of a His-taggedfusion protein of formula (I), wherein the His-tag is the His-tag of SEQID NO:3, wherein A is human serum albumin protein of SEQ ID NO:2, B isthe peptide linker of SEQ ID NO:13, and C is native human MIC-1 of SEQID NO:1.141. A pharmaceutical composition comprising a compound according to anyone of embodiments 1-140 or a pharmaceutically acceptable salt, amide orester thereof, and one or more pharmaceutically acceptable excipients.142. A compound according to any one of embodiments 1-140 for use as amedicament.143. A compound according to any one of embodiments 1-140 for use in theprevention and/or treatment of eating disorders, such as obesity, e.g.by decreasing food intake, reducing body weight, suppressing appetiteand inducing satiety.144. A compound according to any one of embodiments 1-140 for use in theprevention and/or treatment of obesity.145. The use of a compound according to any one of embodiments 1-140 inthe manufacture of a medicament for the treatment of eating disorders,such as obesity, e.g. by decreasing food intake, reducing body weight,suppressing appetite and inducing satiety.146. The use of a compound according to any one of embodiments 1-140 inthe manufacture of a medicament for the treatment of obesity.147. A method of treating or preventing eating disorders, such asobesity, e.g. by decreasing food intake, reducing body weight,suppressing appetite and inducing satiety by administering apharmaceutically active amount of a compound according to any one ofembodiments 1-140.148. A method of treating or preventing obesity by administering apharmaceutically active amount of a compound according to any one ofembodiments 1-140.149. A polynucleotide molecule encoding a compound according to any oneof embodiments 1-140.

EXAMPLES

This experimental part starts with a list of abbreviations, and isfollowed by a section including general methods of preparation,purification and characterisation of the compounds of the invention.Then follows an example relating to the activity and properties of thesefusion proteins (section headed pharmacological methods). The examplesserve to illustrate the invention.

LIST OF ABBREVIATIONS

“Main peak” refers to the peak in a purification chromatogram which hasthe highest UV intensity in milliabsorbance units and which contains thefusion protein.

HPLC is High performance liquid chromatography.

SDS-PAGE is Sodium dodecyl sulfate Polyacrylamide gel electrophoresis.

IMAC is immobilized metal affinity chromatography.

SEC is size exclusion chromatography.

MS is mass spectrometry.

Materials and Methods

General Methods of Preparation

General Expression Method 1: Small Scale Screening and Expression ofFusion Constructs

Expression levels for each construct were determined by transienttransfection of the plasmids into Human Embryonic Kidney (HEK) cells(Expi293F™, Life Technologies™ #A14527) in 2 ml suspension culturesgrown in Expi293™ Expression Medium (Life Technologies™ #A1435101). Theexpi293 cells were grown in disposable 24-well multiwell blocks (Axygen,#P-DW-10 ml-24-C-S) at 37° C., 8% CO₂ and 80% humidity. The shakingspeed was 200 rpm in an Infors Multitron Cell incubator with a 50 mmorbital throw. For each transfection, 2 μg DNA in 100 ul of transfectionmedium (Opti-MEM® I (1×)+GlutaMAX™-I Reduced Serum Medium, LifeTechnologies™ #51985-026) and 5.4 μl ExpiFectamine™ 293 reagent(ExpiFectamine™ 293 Transfection Kit, Life Technologies™ #A14525) intransfection medium were used, according to the manufacturer'sinstructions. 18 hours after transfection, the cultures were fed with 10μl enhancer 1 and 100 μl enhancer 2 (ExpiFectamine™ 293 TransfectionKit, Life Technologies™ #A14525). Approximately 90 hours aftertransfection, the cell cultures were harvested by centrifugation at 4000g for 10 minutes, and the clarified culture medium used for furtheranalysis of protein expression.

The relative expression levels of the constructs were determined byloading clarified cell supernatants directly on SDS-PAGE (Sodium dodecylsulfate Polyacrylamide gel electrophoresis) gels (Novex® NuPAGE® 4-12%Bis-Tris midi protein gels, 26 wells, Life Technologies™ #WG1403BOX)without sample reduction, and the resulting protein bands visualized byCoomassie staining (InstantBlue™, Expedeon #ISBL1L). The productionfeasibility of each fusion protein was assessed by a small scalepurification screen using an immobilized metal affinity chromatography(IMAC) step. The purified protein solutions were visualized by SDS-PAGEand Coomassie staining as described above and the results were used todetermine the cell culture volume needed of each construct to provideenough protein for in vivo assessment of efficacy.

General Expression Method 2: Scale-Up Expression of his-Tagged HSA-MIC-1Fusion Proteins

The plasmids encoding MIC-1 fusion proteins were transformed to OneShot®Top10F″ chemically competent E. coli cells (Life Technologies™#C303003), colonies were grown on Amp/Carb selective agar plates andtransformants used to inoculate liquid Terrific Broth (TB) cultures.After overnight growth, the pelleted E. coli cells were used for largescale plasmid preparations (EndoFree® Plasmid Mega Kit, Qiagen® #12381).

Transient expression was performed by adding plasmid DNA (1 mg/litercell culture) in OptiMEM® transfection medium (50 ml/liter cell culture)to ExpiFectamine™ 293 reagent (2.7 ml/liter cell culture) in OptiMEM®transfection medium (50 ml/liter cell culture), incubating for 20minutes and then adding the transfection mix to the cell culture(expi293F cells at 3×106 cells/ml). 18 hours after transfection, thecultures were fed with enhancers 1 (5 ml/liter cell culture) andenhancer 2 (50 ml/liter cell culture). The expi293 cells were grown in 1liter disposable shaker flasks (Corning #CLS431147) at 37° C., 8% CO₂and 80% humidity. The shaking speed was 110 rpm in an Infors MultitronCell incubator with a 50 mm orbital throw.

Approximately 90 hours after transfection the cultures were harvested bycentrifugation at 4000 g for 10 minutes. The clarified medium wassterile filtered through a 0.22 uM filter before purification.

Purification

Following centrifugation and filtration through a 0.22 μm PES Bottle-topfilter (Techno Plastic Products AG, Switzerland) the clarifiedsupernatant was conditioned for IMAC purification by addition of 200 mLHis-binding buffer (300 mM Sodium Phosphate (NaP), 1.8 M NaCl, 60 mMimidazole, pH 7.5) per liter supernatant.

Using an ÄKTAxpress chromatography system, the conditioned supernatantwas applied at low flowrate to a 5 ml HisTrap Excel column(GE-Healthcare, Sweden) equilibrated in Buffer A (50 mM NaP, 300 mMNaCl, 10 mM Imidazole, pH 7.5) after which low affinity bindingimpurities were eluted with Wash Buffer (50 mM NaP, 300 mM NaCl, 30 mMImidazole, pH 7.5). Bound fusion protein was step eluted with 100%Buffer B (50 mM NaP, 300 mM NaCl, 500 mM Imidazole, pH 7.5) and the mainpeak was collected using the peak detection option of the Unicorn™software and automatically purified further using preparative sizeexclusion chromatography (SEC) in 1×PBS pH 7.4 (Ampliqon) on a HiLoadSuperdex 200 16/600 PG column (GE-Healthcare, Sweden). 1.5 ml fractionswere collected and analyzed by reducing and non-reducing SDS-PAGE usingprecast 4-12% NuPAGE® gels (Life Technologies™). In short the sampleswere mixed with 4×LDS (lithium dodecyl sulphate) sample buffersupplemented with 10× reducing agent when reduction was required. Themixture was heated 5 minutes at 95° C. before loading the SDS-PAGE gels.Novex SeeBlue® plus2 pre-stained Protein standard (Life Technologies™)were run alongside the fractions on SDS-PAGE for size estimation.Protein was visualized using InstantBlue™ stain (Expedeon,Cambridgeshire, UK) according to the manufacturer instructions.

General Expression Method 3: Recombinant Expression of HSA-Linker-MIC-1Fusion Protein

Generation of Vectors for Recombinant Expression of HSA-Linker-MIC-1Fusion Proteins:

A series of CMV promoter-based expression vectors (pTT vectors) weregenerated for transient expression of HSA-linker-MIC-1 fusion proteinsin EXPI293F cells (Life Technologies). The pTT vectors were generatedfor transient protein expression in the HEK293-6E EBNA-based expressionsystem developed by Yves Durocher (Durocher et al. Nucleic AcidResearch, 2002) and can be used for transient expression in the Expi293expression system.

Initially, the gene constructs of each Albumin-linker-MIC-1 fusionprotein variant were ordered in pTT vectors at Genscript with the humanCD33 signal peptide sequence. The plasmids were subsequently transformedinto E. coli for selection and the sequences of the constructs wereverified by DNA sequencing.

Recombinant Expression of Fusion Proteins:

The HSA-linker-MIC-1 fusion proteins were expressed transiently inEXPI293F cells (Life 30 Technologies) by transfection of the pTT-basedexpression vectors according to manufacturer's instructions. Thefollowing procedure describes the generic EXPI293F expression protocol.

Cell Maintenance:

EXPI293F cells were grown in suspension in Expi293™ expression medium(Life Technologies). Cells were cultured in Erlenmeyer shaker flasks inan orbital shaker incubator at 36.5° C., 8% CO2 and 85-125 rpm andmaintained at cell densities between 0.4-4×10E6 cells/mL.

DNA Transfection:

Typically, 30-1000 mL culture volumes were transfected. Separatedilutions of DNA and transfection reagent were initially prepared.Following components were mixed per 1-mL cell culture:

-   -   1. A total of 1 μg vector DNA was diluted in 50 μL Opti-MEM        media (Gibco) and incubated at room temperature (23-25° C.) for        5 min.    -   2. A total of 2.7 μL Expifectamin™ 293 (Life Technologies) was        diluted in 50 μL Opti-MEM media (Gibco) and incubated at room        temperature (23-25° C.) for 5 min.        The two separate dilutions were mixed and incubated at room        temperature (23-25° C.) for 10 min. The DNA-Expifectamin™ 293        mix was added directly to 1 mL EXPI293F cell culture. At the        time of transfection the cell density of the EXPI293F culture        should be 2.8-3.2×10E6 cells/mL. The transfected cell cultures        were incubated in an orbital shaker incubator at 36.5° C., 8%        CO2 and 85-125 rpm. 18 hrs post transfection; 5 uL Expifectamin™        293 Transfection Enhancer 1 and 50 uL Expifectamin™ 293        Transfection Enhancer 2 were added per 1-mL culture. 5 days post        transfection; the cell culture supernatants were harvested by        centrifugation, followed by filtration through a 0.22 μm PES        filter unit (Corning).

Purification

The fusion proteins were captured on a Gigacap column (ion exchange) atpH 8 (neutral pH) and eluted with an increase in salt (sodium sulphate)concentration using a stepwise gradient. The eluted protein was eitherconcentrated on Amicon Ultra centrifugal filters with a MWCO of 10 kDaor not, depending on the concentration in the capture pool. The analoguewas finally purified on a HiLoad Superdex200 16/60 or 26/60 prep gradecolumn using a PBS buffer.

General Methods of Detection and Characterisation

MS Analysis

Intact mass of the purified combined fusion protein was analysed usingThermo-Dionex Ultimate3000™ HPLC (Thermo Fisher Scientific) coupled to aMaxis Impact™ ESI-Q-OTOF mass spectrometer (Bruker Daltonics). Solventswere A: Water with 0.1% Formic Acid (v/v) and B: Acetonitrile with 0.08%Formic acid (v/v). The sample was desalted online on a Waters Acquity™BEH300 C4 1.7 μm 1.0×100 mm column (Waters) for 2 minutes in 10% B, 0.2ml/min and eluted by a 8 minute linear gradient from 10% B to 90% Bsolvent at 0.2 ml/min.

Absorbance at 215 nm (Abs215) and m/z spectra in the range m/z 300 to3000 were recorded. Obtained data was analysed using the DataAnalysis4.1 software (Bruker Daltonics). Averaged m/z spectra were deconvolutedusing Maximum Entropy deconvolution.

Peptide mass mapping was performed to verify correct linker sequencesand was done using methods know to persons skilled in the art. In short,purified proteins were subjected to tryptic digestion using a methodadopted from “In solution tryptic digest and guanidation kit”, Pierceproduct nr. 89895. Peptide mass mapping to allow identification andverification of the correct linker sequence in the fusion proteins wasdone using the Data analysis software (Bruker Daltonics) to extractexperimental determined masses of peptides and the Biotools software(Biotools) for matching experimental masses against the calculatedmasses derived from the expected fusion protein sequences according tothe manufacturer's instructions. In general, variable modifications wereset to “Oxidation (M)” and “Carbamidomethyl (C)” and the mass tolerancewas set to 20 ppm and MS/MS tolerance to 50 mmu.

Chemiluminescent Nitrogen Detection (CLND) coupled to a standard HPLCwas used to determine the protein concentration essentially as describedelsewhere (eg. Bizanek, R.; Manes, 3. D.; Fujinari, E. M.Chemiluminescent nitrogen detection as a new technique for purityassessment of synthetic peptides separated by reversed-phase HPLC. Pept.Res. 1996, 9 (1), 40-44).

Example 1: Expression and Purification of the Compounds of the Invention

The different plasmids encoding the fusion protein variants depicted inTable 1 were designed with differences in the linker sequence betweenthe human serum albumin part and the MIC-1 part.

TABLE 1 List of HSA MIC-1 fusion proteins. Compounds referred to in thetable has human serum albumin or a human serum albumin variant in theN-terminal, a linker sequence as indicated with an amino acid sequenceand wild type human MIC-1 or a MIC-1 functional variant in theC-terminal (see also FIG. 1). An N-terminal His6 tag (SEQ ID NO: 3) wasincluded for some constructs to facilitate IMAC purification. PeptideHuman General map serum expression (sequence N-terminal albumin LinkerMIC-1 method coverage, Compound His-tag (HSA) sequence Protein used (%)1 SEQ ID SEQ ID No linker SEQ ID NO: 1 2 53.8 NO: 3 NO: 2 2 SEQ ID SEQID SEQ ID NO: 4 SEQ ID NO: 1 2 68.3 NO: 3 NO: 2 3 SEQ ID SEQ ID SEQ IDNO: 5 SEQ ID NO: 1 2 82.8 NO: 3 NO: 2 4 SEQ ID SEQ ID SEQ ID NO: 6 SEQID NO: 1 2 32.0 NO: 3 NO: 2 5 SEQ ID SEQ ID SEQ ID NO: 10 SEQ ID NO: 1 260.5 NO: 3 NO: 2 6 SEQ ID SEQ ID SEQ ID NO: 8 SEQ ID NO: 1 2 70.0 NO: 3NO: 2 7 SEQ ID SEQ ID SEQ ID NO: 9 SEQ ID NO: 1 2 53.6 NO: 3 NO: 2 8 SEQID SEQ ID SEQ ID NO: 7 SEQ ID NO: 1 2 62.9 NO: 3 NO: 2 9 SEQ ID SEQ IDSEQ ID NO: 11 SEQ ID NO: 1 2 69.5 NO: 3 NO: 2 10 SEQ ID SEQ ID SEQ IDNO: 12 SEQ ID NO: 1 2 66.3 NO: 3 NO: 2 11 SEQ ID SEQ ID SEQ ID NO: 13SEQ ID NO: 1 2 71.6 NO: 3 NO: 2 12 SEQ ID SEQ ID SEQ ID NO: 24 SEQ IDNO: 1 2 49.4 NO: 3 NO: 2 13 SEQ ID SEQ ID SEQ ID NO: 25 SEQ ID NO: 1 262.5 NO: 3 NO: 2 14 SEQ ID SEQ ID SEQ ID NO: 26 SEQ ID NO: 1 2 64.1 NO:3 NO: 2 15 SEQ ID SEQ ID SEQ ID NO: 27 SEQ ID NO: 1 2 75.5 NO: 3 NO: 216 SEQ ID SEQ ID SEQ ID NO: 28 SEQ ID NO: 1 2 64.8 NO: 3 NO: 2 17 SEQ IDSEQ ID SEQ ID NO: 29 SEQ ID NO: 1 2 31.0 NO: 3 NO: 2 18 SEQ ID SEQ IDSEQ ID NO: 30 SEQ ID NO: 1 2 73.1 NO: 3 NO: 2 19 SEQ ID SEQ ID SEQ IDNO: 31 SEQ ID NO: 1 2 84.7 NO: 3 NO: 2 20 SEQ ID SEQ ID SEQ ID NO: 32SEQ ID NO: 1 2 78.3 NO: 3 NO: 2 21 SEQ ID SEQ ID SEQ ID NO: 33 SEQ IDNO: 1 2 74.0 NO: 3 NO: 2 22 SEQ ID SEQ ID SEQ ID NO: 34 SEQ ID NO: 1 281.8 NO: 3 NO: 2 23 no His- SEQ ID SEQ ID NO: 9 SEQ ID NO: 1 3 75.1 tagNO: 2 24 no His- SEQ ID SEQ ID NO: 9 SEQ ID NO: 1 3 79.3 tag NO: 23 25no His- SEQ ID SEQ ID NO: 9 SEQ ID NO: 14 3 71.0 tag NO: 23 26 no His-SEQ ID SEQ ID NO: 9 SEQ ID NO: 15 3 73.8 tag NO: 23 27 SEQ ID SEQ ID SEQID NO: 9 SEQ ID NO: 16 2 87.0 NO: 3 NO: 23 28 SEQ ID SEQ ID SEQ ID NO: 9SEQ ID NO: 17 2 86.5 NO: 3 NO: 23 29 SEQ ID SEQ ID SEQ ID NO: 9 SEQ IDNO: 18 2 84.3 NO: 3 NO: 23 30 SEQ ID SEQ ID SEQ ID NO: 9 SEQ ID NO: 19 282.2 NO: 3 NO: 23 31 SEQ ID SEQ ID SEQ ID NO: 9 SEQ ID NO: 20 2 91.1 NO:3 NO: 23 32 SEQ ID SEQ ID SEQ ID NO: 9 SEQ ID NO: 21 2 83.6 NO: 3 NO: 2333 SEQ ID SEQ ID SEQ ID NO: 35 SEQ ID NO: 1 2 62.6 NO: 3 NO: 2 34 SEQ IDSEQ ID SEQ ID NO: 36 SEQ ID NO: 1 2 75.1 NO: 3 NO: 2 35 SEQ ID SEQ IDSEQ ID NO: 37 SEQ ID NO: 1 2 79.6 NO: 3 NO: 2 36 SEQ ID SEQ ID SEQ IDNO: 38 SEQ ID NO: 1 2 74.1 NO: 3 NO: 2 37 SEQ ID SEQ ID SEQ ID NO: 9 SEQID NO: 22 2 76.4 NO: 3 NO: 23

Some fusion proteins comprise wild type human MIC-1 (SEQ ID NO:1),others MIC-1 variants (SEQ ID NO:14-SEQ ID NO:23). Some fusion proteinscomprises human wild type human serum albumin (SEQ ID NO:2), others HSAC34A, a human serum albumin variant wherein the cysteine residue atposition 34 of the wild type human serum albumin amino acid sequence hasbeen replaced with alanine (SEQ ID NO:23). Plasmids were generated bywell-known recombinant DNA technology methods (obtained from GenScriptInc). Constructs were designed with or without a N-terminal His tagsequence (SEQ ID NO:3). Constructs with His tag allows directpurification using immobilized affinity chromatography (IMAC), whereasother means of purification was used for purification of non-His taggedconstructs. Since the His-tag is placed in the very N-terminal of humanserum albumin it does not affect neither the efficacy of the MIC-1fusion proteins, nor the binding of human serum albumin to Fc NeonatalReceptor and the half-life extending effect of human serum albumin, whenused as a fusion partner. The linker sequence is given in Table 2.

TABLE 2  List of peptide linkers with corresponding SEQ ID NO and amino acid sequence. SEQ ID NO Linker sequenceSEQ ID NO: 4 EAAEAAE SEQ ID NO: 5 EEEAEEEAEEEAEEEAEEE SEQ ID NO: 6GGSSSGSGGSGGSGSGGSGGSGS SEQ ID NO: 7 DDADDADDADDADDADDAD SEQ ID NO: 8KAAKAAKAAKAAKAAKAAK SEQ ID NO: 9 GGSSEAAEAAEAAEAAEAAEAAE SEQ ID NO: 10DAADAADAADAADAADAAD SEQ ID NO: 11 EAAEAAEAAEAAEAAEAAE SEQ ID NO: 12EAAEAAEAAEAAEAAEAAEAAEAAEAAEAAE SEQ ID NO: 13GGSSEAAEAAEAAEAAEAAEAAEAAEAAEAAEAAE SEQ ID NO: 24 AAEGEEEAESEQ ID NO: 25 GGSSSGS SEQ ID NO: 26 PTPTPTP SEQ ID NO: 27GGSSEEEAEEEAEEEAEEEAEEE SEQ ID NO: 28 GGSSSGSGGSGGSGSGGSGGSGSGSGGSGGSSEQ ID N0: 29 GGSSPTPTPTPTPTPTPTPTPTP SEQ ID NO: 30PTPTPTPTPTPTPTPTPTPTPTPTPTPTPTP SEQ ID NO: 31 QAAAQAAAQAAAQAAAQAAAQAAAQSEQ ID N0: 32 QAAQAAQAAQAAQAAQAAQ SEQ ID NO: 33 EAAAEAAAEAAAEAAAEAAAESEQ ID NO: 34 DAAADAAADAAADAAADAAAD SEQ ID NO: 35GGSSEAAAEAAAEAAAEAAAEAAAE SEQ ID NO: 36 EAAAEAAAEAAAEAAAEAAAEAAAESEQ ID NO: 37 DAAADAAADAAADAAADAAADAAAD SEQ ID NO: 38GGSSEAAAEAAAEAAAEAAAEAAAEAAAE

TABLE 3 List of MIC-1 variants with corresponding SEQ ID NO. SEQ ID NOMIC-1 variants SEQ ID NO: 1 Wild type human MIC-1 (hMIC-1) SEQ ID NO: 14N3S hMIC-1 SEQ ID NO: 15 R2A, N3E hMIC-1 SEQ ID NO: 16 N3E hMIC-1 SEQ IDNO: 17 N3A hMIC-1 SEQ ID NO: 18 N3P hMIC-1 SEQ ID NO: 19 N3T hMIC-1 SEQID NO: 20 N3G hMIC-1 SEQ ID NO: 21 N3Q hMIC-1 SEQ ID NO: 22 N3D hMIC-1

TABLE 4 List of human serum albumin (HSA) variants with correspondingSEQ ID NO. SEQ ID NO Human serum albumin variants SEQ ID NO: 2  Wildtype HSA SEQ ID NO: 23 C34A HSA

As a representative example, large scale production of Compound no. 7was performed by transient expression in Expi293F cells as described inmaterials and methods section. Briefly, 200 μg plasmid DNA was added to10 ml of Opti-MEM® transfection medium and 540 μl ExpiFectamine™ 293reagent was added to 10 ml of Opti-MEM® transfection medium. The twosolutions were combined to form a transfection mix. After 20 minutesincubation, the transfection mix was added to 200 ml of expi293F cellculture with a cell density of 3×106 cells/ml. 18 hours aftertransfection, the cultures were fed with 1 ml of enhancer 1 and 10 ml ofenhancer 2. Approximately 90 hours after transfection the culture washarvested by centrifugation at 4000 g for 10 minutes. The clarifiedmedium was sterile filtered through a 0.22 uM filter beforepurification.

To examine the in vivo effect of fusing a human serum albumin moleculeto the N-terminus of the MIC-1 protein by variable linkers the expressedmolecule were purified using the method described above. Compound no. 7was successfully purified using automated immobilized metal ionchromatography coupled to size exclusion. Two major peaks within thetotal volume of the SEC column were fractioned and analysed. The firstpeak eluted at the void of the column and non-reducing SDS-PAGEconfirmed the aggregated state of the eluted protein. The main peakpartially overlapped with the aggregate peak. Therefore, not allfractions representing the entire main peak were included in the pool.Non-reducing SDS-PAGE of the pooled fractions resulted in a single bandwhich migrated as a ˜120 kDa protein. To verify the dimeric structure ofthe molecule, intact mass spectrometry (MS) was performed. Deconvolutionof the averaged mass spectra resulted in the average mass 164140 Da. Thecalculated molecular weight of Compound no. 7 is 163820 Da. Thus, intactMS analysis shows that the purified molecule is in its dimeric form, butpotentially carries several post translational modifications (e.g.oxidations, deamidations etc.). To further characterise the constructs,peptide mass mapping strategies were deployed for characterisation.HSA-MIC-1 fusion proteins expressed in the mammalian host cells,produced varying degree of Cys34 cysteinylation as described previously(Kleinova A, et al., Rapid Commun. Mass Spectrom, 2005; 19: 2965-2973.).In addition, it was found that other causes of heterogeniety was linkedto the Asn in position 3 of the MIC-1 sequence, which was found highlylabile, since it readily deamidated to Asp or isoAsp.

Pharmacological Methods

Example 2: Effect of Fusions Proteins of the Invention on Food Intake inLean Sprague Dawley Rats

The purpose of this example is to test the efficacy of the compounds invivo. The in vivo efficacy of the compounds of the invention wasmeasured in 250 g-300 g male non-obese Sprague Dawley rats. Animals wereinjected once with a dose of 4 nmol/kg body weight. Compounds wereadministrate subcutaneously (1 ml/kg) in a physiological isotonicphosphate buffered saline (PBS) solution (137 mM NaCL; 2.7 mM KCl; 10 mMNa₂HPO₄; 1.8 mM KH₂PO₄). In some cases the buffered saline solution alsocontained 500 ppm of polysorbate 80. Wild-type human MIC-1 was includedas a reference compound and was injected once daily during the studywith a dose of 8 nmol/kg body weight. Wild-type hMIC-1 was administeredsubcutaneously (1 ml/kg) in an acidic isotonic buffered solution (pH4.0; 5 mM acetate, 2.25% glycerol, 70 ppm polysorbate 20).

Changes in food intake were measured either by an automatic foodmonitoring system (BioDAQ or HM-2) or by measuring the reduction in foodpellets in the cage feeding tray manually over a 24 hr period of time.Animals were single housed in the BioDAQ system and housed 3 per cage inthe HM-2 system. Animals were in the latter system chip-marked priorstudy start in order for the HM-2 system to collect individual measuresof food intake. Each compound was tested in n=4-8 animals in one or moreexperiments. Animals were acclimatized for at least for 7 days in theexperimental set up prior to study start. Collected data are expressedas daily food intake (24 hour food intake) measured from the onset ofeach daily 12 hour dark phase to the following dark phase. Daily changesin food intake in response to administered compound were in most studiescalculated by subtracting the average daily food intake of the treatmentgroup from the average daily food intake of the vehicle group. In a fewstudies daily changes in food intake in response to administeredcompound were calculated by subtracting the daily average food intakeduring the intervention from the average daily food intake of the dayprior to study start. Changes were considered significant if p<0.1 usinga student's t-test (two-tailed).

Several amino acid linkers between the human serum albumin part and theMIC-1 part were explored. The linkers were characterised by havingdifferent lengths, charges or structural motifs (eg. Pro-rich linkers,linkers with predicted alpha-helical propensities comprising Glu/Asp andAla or typical Gly/Ser containing linkers conferring linkerflexibility). The linker variants were evaluated and compared on basisof max efficacy, duration of biological effect and accumulated efficacyas described above.

The inventors surprisingly found that the absence of a linker or peptidelinkers with a size below 10 amino acids between human serum albumin andMIC-1 resulted in compounds with limited or no significant biologicalefficacy (compounds 1, 2, 12, 13 and 14 (table 5)).

In contrast, linkers with a size of 10 or more amino acids positivelyinfluenced the biological efficacy of the fusion protein. In the presentinvention it was also found that variation in the linker amino acidcomposition and sequence also significantly influenced the biologicalefficacy of the HSA MIC-1 fusion proteins. The inventors surprisinglyfound that a specific combination of medium sized linkers of about 20amino acids comprising repeats of an acidic residue (Glu or Asp)followed by at least two non-polar residues such as Ala resulted inincreased biological efficacy of the HSA-MIC-1 fusion protein whencompared to flexible linkers of identical size comprising Gly and Serresidues, which are normally used as linkers for separating domains offusion proteins (Table 3, Compound no. 7 or 9 compared with Compound no.4).

TABLE 5 Effect of a single dose (4 nmol/kg) of comparative HSA-MIC-1fusion proteins on daily food intake in lean SD rats. Data are expressedin 3 ways, 1) maximum efficacy which is the greatest significant (p <0.10) reduction in 24 hours food intake recorded over the study period,2) Accumulated efficacy which is the sum of significant (p < 0.10)reductions in 24 hours food intake compared with vehicle and 3) Durationof effect which is the number of days with a significant (p < 0.1)reductions in food intake compared with vehicle. Wild-type human MIC-1is included for comparison and was administered once daily for 7 days (8nmol/kg). Length of linker (number of Maximum Accumulated Duration ofCompound amino acids) efficacy efficacy effect wt MIC-1 0 37 n/a 1 (SEQID NO: 1) 1 0 0 0 0 2 7 11 11 1 12 9 11 8 1 13 7 −12 −12 1 14 7 15 1 2 819 23 66 3 3 19 30 74 4 15 23 20 38 2 4 23 28 72 3 16 31 22 56 3 6 19 1834 2 17 23 30 78 3 18 31 23 23 1 19 25 20 51 3 20 19 30 83 3

TABLE 6 Effect of a single dose (4 nmol/kg) of HSA-MIC-1 fusion proteinsof the invention having varying linker length on daily food intake inlean SD rats. Data are expressed in 3 ways, 1) maximum efficacy which isthe greatest significant (p < 0.10) reduction in 24 hours food intakerecorded over the study period, 2) Accumulated efficacy which is the sumof significant (p < 0.10) reductions in 24 hours food intake comparedwith vehicle and 3) Duration of effect which is the number of days witha significant (p < 0.1) reductions in food intake compared with vehicle.Wild-type human MIC-1 is included for comparison and was administeredonce daily for 7 days (8 nmol/kg). Length of linker (number of MaximumAccumulated Duration of Compound amino acids) efficacy efficacy effectwt MIC-1 0 37 n/a 1 (SEQ ID NO: 1) 5 19 42 183 6 g 19 35 86 4 21 21 3380 3 22 21 39 183 6 7 23 39 167 6 23 23 31 98 4 33 25 37 154 5 34 25 31135 5 35 25 38 149 5 36 29 39 169 6 10 31 31 91 4 11 35 25 60 3

More in particular, the inventors surprisingly found that medium sizedlinkers of about 10-35 amino acids comprising repeats of an acidicresidue (Glu or Asp) followed by at least two non-polar residues such asAla showed favorable biological efficacy of the HSA-MIC-1 fusionprotein, when compared to flexible linkers of identical size comprisingGly and Ser residues or rigid Pro containing linkers (compound 7 (table6) compared with compounds 4 and 17 (table 5)). Similar observationswere done for longer linkers above 30 aa (compound 10 (table 6) comparedwith compound 16 or 18 (table 5)). Substitutions of Ala with acidicresidues in each repeat negatively affected the maximum efficacy and/oraccumulated efficacy (e.g. compound 5 (table 6) compared to compound 8(table 5)). Surprisingly, it was found that substitution of the acidicresidues of linkers containing repeats of Glu-Ala-Ala or Asp-Ala-Alawith a basic Lys residue resulted in a clear decrease in biologicalefficacy and accumulated food intake demonstrating that difference inefficacy can result from small changes in the linker sequence (Compoundno. 5 and 9 (table 6) compared to compound 6 (table 5)).

Thus, the present invention demonstrates that HSA MIC-1 fusion proteinswith certain linkers results in higher maximum efficacy and accumulatedefficacy as well as longer duration of the fusion protein.

TABLE 7 Effect of a single dose (4 nmol/kg) of HSA-MIC-1 fusion proteinsof the invention all having a linker of SEQ ID NO: 9, and comprising aMIC-1 variant and/or human serum albumin variant on daily food intake inlean SD rats. Data are expressed in 3 ways, 1) maximum efficacy which isthe greatest significant (p < 0.10) reduction in 24 hours food intakerecorded over the study period, 2) Accumulated efficacy which is the sumof significant (p < 0.10) reductions in 24 hours food intake comparedwith vehicle and 3) Duration of effect which is the number of days witha significant (p < 0.1) reductions in food intake compared with vehicle.Wild-type hMIC-1 is included for comparison and was administered oncedaily for 7 days (8 nmol/kg). Max- Accum- Dura- MIC-1/ imum ulated tionof Compound HSA variant MIC-1 variant efficacy efficacy effect wt MIC-1— — 37 n/a 1 (SEQ ID NO: 1) 24 SEQ ID NO: 23 SEQ ID NO: 1 29 69 3 25 SEQID NO: 23 SEQ ID NO: 14 38 127 5 26 SEQ ID NO: 23 SEQ ID NO: 15 30 114 527 SEQ ID NO: 23 SEQ ID NO: 16 31 87 3 28 SEQ ID NO: 23 SEQ ID NO: 17 3189 4 29 SEQ ID NO: 23 SEQ ID NO: 18 38 78 3 30 SEQ ID NO: 23 SEQ ID NO:19 22 54 3 31 SEQ ID NO: 23 SEQ ID NO: 20 30 74 3 32 SEQ ID NO: 23 SEQID NO: 21 34 113 4 37 SEQ ID NO: 23 SEQ ID NO: 22 44 173 5

HSA MIC-1 fusions proteins, all with a linker of SEQ ID NO:9, andcomprising the MIC-1 variants and/or the human serum albumin variants oftables 3 and 4, respectively, were prepared and tested to investigate ifthese changes in the MIC-1 part and/or the human serum albumin part hadan effect on the efficacy of the fusion proteins. As can be seen intable 7, all the fusions proteins were found to significantly reducefood intake for 3-5 days in response to a single injection of 4 nmol/kg.

Example 3: Effect of Fusions Proteins of the Invention on Food Intake inDIO Sprague Dawley Rats

DIO rats were used to further study compounds tested in lean rats.Obesity was induced by placing eight-week-old animals on a specialresearch diet (Research Diets, D12451) where 45% of the energy contentis derived from fat. Animals typically reached a body weight of 500-600g before study initiation. Animals were injected once with a dose of 4nmol/kg body weight. Compounds were administered subcutaneously (1ml/kg) in a physiological isotonic phosphate buffered saline (PBS)solution (137 mM NaCL; 2.7 mM KCl; 10 mM Na₂HPO₄; 1.8 mM KH₂PO₄). Insome cases the buffered saline solution also contained 500 ppm ofpolysorbate 80.

Changes in food intake were measured by an automatic food monitoringsystem (BioDAQ or HM-2). Animals were single housed in the BioDAQ systemand housed 3 per cage in the in the HM-2 system. Animals were in thelatter system chip-marked prior to study start in order for the HM-2system to collect individual measures of food intake. Each compound wastested in n=4-8 animals in one or more experiments. Animals wereacclimatized for at least 7 days in the experimental set up prior tostudy start. Collected food intake data are expressed as daily foodintake (24 hour food intake) measure from the onset of each daily 12hour dark phase to the following dark phase. Daily changes in foodintake in response to administered compound were calculated bysubtracting the average daily food intake of the treatment group fromthe average daily food intake of the vehicle group. Changes wereconsidered significant if p<0.1 using a student's t-test (two-tailed).

The HSA MIC-1 fusion proteins tested all displayed good efficacy in DIOrats. When comparing compound 7 and 23, it is apparent that the His-tag(SEQ ID NO:3) does not affect the efficacy or duration of effect of thefusion proteins.

TABLE 8 Effect of a single dose (4 nmol/kg) of HSA-MIC-1 analogues onbody weight and daily food intake in obese SD rats. Data are expressedin 4 ways, 1) maximum efficacy which is the greatest significant (p <0.10) reduction in 24 hours food intake recorded over the study period,2) Accumulated efficacy which is the sum of significant (p < 0.10)reductions in 24 hours food intake compared with vehicle and 3) Durationof effect which is the number of days with a significant (p < 0.1)reductions in food intake compared with vehicle, 4) Differences in bodyweight at day 7 compared to the vehicle group. Maximum AccumulatedDuration of % body weight Compound efficacy efficacy effect difference 561 309 6 −6.7 7 68 369 6 −8.9 9 56 331 7 −7.1 23 70 355 7 −8.8 24 62 3316 −10 26 64 385 7 −9.6

Example 4: Pharmacokinetic Evaluation of MIC-1 Compounds in Lean SpragueDawley Rats

The purpose of this study is to determine the half-life in vivo of theHSA MIC-1 fusion proteins after intravenous administration to leanSprague Dawley rats, i.e. the prolongation of their time in the bloodcirculation and thereby their time of action. This is done in apharmacokinetic (PK) study, where the terminal half-life of the fusionprotein in question is determined. By terminal half-life is generallymeant the period of time it takes to halve a certain plasmaconcentration, measured after the initial distribution phase.

The in vivo half-life was measured in 300 g-500 g lean SD rats byinjecting the compound into the tail vein followed by collection ofblood plasma samples at various time points for exposure analysis.Compounds (0.5 nmol/kg body weight) were administered intravenously (1ml/kg) in a physiologically isotonic phosphate buffered saline (PBS)solution (140 mM NaCL; 2.7 mM KCl; 8.05 mM Na₂HPO₄; 1.96 mM KH₂PO₄, 500ppm polysorbate 80). Blood samples were collected from the tongue attime −30, 30, 60, 240 and 420 minutes and 24, 30, 48, 72, 96, 120, 168,216, 264 and 360/384 hours. 200 μl of blood was collected into EDTAtubes and stored on ice for up to 20 minutes. Plasma samples weregenerated by centrifuging blood samples for 5 minutes at 10000 G at 4°C. The sample was subsequent pipetted into Micronic tubes on dry ice,and kept at −20° C. until analysed for plasma concentration of therespective MIC-1 compound using LOCI or a similar antibody based assaysuch as ELISA. The individual plasma concentration-time profiles wereanalysed by a non-compartmental model in Phoenix v. 6.2 or 6.3 software(Pharsight Inc., Mountain View, Calif., USA), and the resulting terminalhalf-lives determined.

TABLE 9 Pharmacokinetic profile of MIC-1 compounds in lean SD rats (0.5nmol/kg) with intravenous tail vein dosing. Data are expressed as thehalf-life (T½). Compound intravenous T½ (hours) wt hMIC-1 (SEQ ID NO: 1)1.9 1 38 2 32 3 27 4 29 5 27 6 27 7 36 8 19 9 37 10 2 13 2 14 41 16 3 1845 20 35 21 39 23 46 24 42 25 38 26 49 33 36 35 31 36 37

A correlation between the length of the linker (i.e—the number of aminoacids in the linker) and the T½ in lean rat was analysed using a Pearsoncorrelation analysis. The Spearman correlation coefficient was −0.0365suggesting no significant linear relationship between the linker lengthand T½. An implication of this analysis is that the biological efficacyof the fusion proteins is not a function of the in vivo half-life of thefusion proteins.

While certain features of the invention have been illustrated anddescribed herein, many modifications, substitutions, changes, andequivalents will now occur to those of ordinary skill in the art. It is,therefore, to be understood that the appended claims are intended tocover all such modifications and changes as fall within the true spiritof the invention.

The invention claimed is:
 1. A fusion protein of formula (I):A-B-C  (I), wherein A is human serum albumin or a functional variantthereof; B is a peptide linker 10 to 50 amino acids in length andcomprising the formula [X-Y_(m)]_(n), wherein X is Asp or Glu; Y is Ala;m is from 2 to 4; and n is from 2 to 10; and C is a MIC-1 protein or ananalogue thereof; wherein the C-terminus of the human serum albumin orfunctional variant thereof is fused to the N-terminus of the peptidelinker; and wherein the C-terminus of the peptide linker is fused to theN-terminus of the MIC-1 protein or analogue thereof.
 2. A compoundcomprising a homodimer of a fusion protein of formula (I):A-B-C  (I), wherein A is human serum albumin or a functional variantthereof; B is a peptide linker 10 to 50 amino acids in length andcomprising the formula [X-Y_(m)]_(n), wherein X is Asp or Glu; Y is Ala;m is from 2 to 4; and n is from 2 to 10; and C is a MIC-1 protein or ananalogue thereof; wherein the C-terminus of the human serum albumin orfunctional variant thereof is fused to the N-terminus of the peptidelinker; wherein the C-terminus of the peptide linker is fused to theN-terminus of the MIC-1 protein or analogue thereof; and wherein thehomodimer comprises an interchain disulphide bridge between the MIC-1protein or analogue thereof of each fusion protein.
 3. A pharmaceuticalcomposition comprising a fusion protein of formula (I):A-B-C  (I), wherein A is human serum albumin or a functional variantthereof; B is a peptide linker 10 to 50 amino acids in length andcomprising the formula [X-Y_(m)]_(n), wherein X is Asp or Glu; Y is Ala;m is from 2 to 4; and n is from 2 to 10; and C is a MIC-1 protein or ananalogue thereof; wherein the C-terminus of the human serum albumin orfunctional variant thereof is fused to the N-terminus of the peptidelinker; and wherein the C-terminus of the peptide linker is fused to theN-terminus of the MIC-1 protein or analogue thereof; or apharmaceutically acceptable salt, amide, or ester thereof.
 4. Thepharmaceutical composition according to claim 3, further comprising ahomodimer of the fusion protein, wherein the homodimer comprises aninterchain disulphide bridge between the MIC-1 analog of each fusionprotein.
 5. A method of treating obesity by administering apharmaceutically active amount of a fusion protein of formula (I):A-B-C  (I), wherein A is human serum albumin or a functional variantthereof; B is a peptide linker 10 to 50 amino acids in length andcomprising the formula [X-Y_(m)]_(n), wherein X is Asp or Glu; Y is Ala;m is from 2 to 4; and n is from 2 to 10; and C is a MIC-1 protein or ananalogue thereof; wherein the C-terminus of the human serum albumin orfunctional variant thereof is fused to the N-terminus of the peptidelinker; and wherein the C-terminus of the peptide linker is fused to theN-terminus of the MIC-1 protein or analogue thereof.
 6. The methodaccording to claim 5, further comprising administering a homodimer ofthe fusion protein, wherein the homodimer comprises an interchaindisulphide bridge between the MIC-1 analog of each fusion protein. 7.The fusion protein according to claim 1, wherein A is selected from thegroup consisting of SEQ ID NO:2 and SEQ ID NO:23.
 8. The fusion proteinaccording to claim 1, wherein B is selected from the group consisting ofSEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQID NO:13, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ IDNO:37, and SEQ ID NO:38.
 9. The fusion protein according to claim 1,wherein C is selected from the group consisting of SEQ ID NO:1, SEQ IDNO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:21, and SEQ ID NO:22.
 10. The fusion protein according to claim 1,wherein A is selected from the group consisting of SEQ ID NO:2 and SEQID NO:23; wherein B is selected from the group consisting of SEQ IDNO:4, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ IDNO:37, and SEQ ID NO:38; and wherein C is selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQID NO:19, SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22.